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Research Article
Access to Photostability-Enhanced Unnatural Base Pairs via Local Structural Modifications
通过局部结构修饰获得光稳定性增强的非天然碱基对Click to copy article link
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- Honglei WangHonglei WangHenan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, ChinaMore by Honglei Wang
- Luying WangLuying WangHenan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, ChinaMore by Luying Wang
- Nana MaNana MaHenan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, ChinaMore by Nana Ma
- Wuyuan ZhuWuyuan ZhuHenan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, ChinaMore by Wuyuan Zhu
- Bianbian HuoBianbian HuoHenan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, ChinaMore by Bianbian Huo
- Anlian ZhuAnlian ZhuHenan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, ChinaMore by Anlian Zhu
- Lingjun Li*Lingjun Li*Email: lingjunlee@htu.edu.cn. Tel: +863733326335. Fax: +863733326335.Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, ChinaMore by Lingjun Li
Abstract 抽象
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Completing the storage and retrieval of increased genetic information in vivo and producing therapeutic proteins have been achieved by the unnatural base pair dNaM-dTPT3. Up to now, some biological and chemical approaches are implemented to improve the semi-synthetic organism (SSO). However, the photosensitivity of this pair, suggested as a potential threat to the healthy growth of cells, is still a problem to solve. Hence, we designed and synthesized a panel of TPT3 analogues with the basic structural skeletons of TPT3 but modified thiophene rings at variant sites to improve the photostability of unnatural base pairs. A comprehensive screening strategy, including photosensitivity tests, kinetic experiments, and replication in vitro by PCR and in vivo by amplification, was implemented. A new pair, dNaM-dTAT1, which had almost equally high efficiency and fidelity with the dNaM-dTPT3 pair itself both in vivo and in vitro, was proven to be more photostable and thermostable and less toxic to E. coli cells. The discovery of dNaM-dTAT1 represents our first progress for the optimization of this type of bases toward more photostable properties; our data also suggest that less photosensitive unnatural base pairs will be beneficial to build a healthier cellular replication system.
非天然碱基对 dNaM-dTPT3 完成了体内增加遗传信息的存储和检索并产生治疗性蛋白质。到目前为止，已经实施了一些生物和化学方法来改进半合成生物体 （SSO）。然而，这对光敏性被认为是对细胞健康生长的潜在威胁，仍然是一个需要解决的问题。因此，我们设计并合成了一组具有 TPT3 基本结构骨架的 TPT3 类似物，但在变体位点修饰了噻吩环，以提高非天然碱基对的光稳定性。实施了全面的筛选策略，包括光敏性测试、动力学实验以及通过 PCR 在体外和通过扩增在体内进行复制。一种新的 dNaM-dTAT1 对在体内和体外都与 dNaM-dTPT3 对本身具有几乎相同的效率和保真度，被证明具有更高的光稳定性和热稳定性，并且对大肠杆菌细胞的毒性更小。dNaM-dTAT1 的发现代表了我们在优化此类碱基以获得更多光稳定性方面的首次进展;我们的数据还表明，光敏感度较低的非天然碱基对将有利于构建更健康的细胞复制系统。
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Copyright © 2021 American Chemical Society
版权所有 © 2021 美国化学会
版权所有 © 2021 美国化学会
Developments of unnatural base pairs (UBPs) to expand the genetic alphabet offers an opportunity to increase the information storage capacity of DNA, (1−3) simultaneously providing a platform for promising biotechnological applications including site-specific modifications of nucleic acids, (4,5) SELEX for high-affinity aptamers, (6−8) and production of therapeutic proteins bearing noncanonical amino acids. (9−12) However, it is always a challenging task to discover new unnatural bases possessing alien structural scaffolds from natural bases but having desirable chemical and biological properties. From the 1960s, researchers have explored hundreds of unnatural base pairs by variant design strategies such as different hydrogen-bonding patterns, hydrophobic packing interactions, and shape complementarities, (13−15) which representatively resulted in three types of pairs, TPT3-NaM, (16,17) Ds-Px, (18) and P-Z. (19,20) These pairs have high efficiency, fidelity, and bio-orthogonal functions. Especially, TPT3-NaM can be recently utilized in a semi-synthetic organism (SSO) to complete the storage and retrieval of increased genetic information. (9,12,17) Although the discovery of TPT3-NaM represented a milestone for applications of unnatural base pairs in synthetic biology, some drawbacks of this pair are also found in view of its further applications. The photosensitivity of the TPT3-NaM pair has been pointed out as a potential threat to the healthy growth of cells. (21−23) Typically, the strong near-visible light absorption of TPT3 can cause a dramatic increase of ROS in cellular cultures and thus lead to irreversible destruction of cells. To our best knowledge, the methods to improve the photostability of unnatural base pairs are still stranded.
开发非天然碱基对 （UBP） 以扩展遗传字母表为增加 DNA 的信息存储容量提供了机会，（1-3） 同时为有前途的生物技术应用提供了一个平台，包括核酸的位点特异性修饰，（4,5） 高亲和力适配体的 SELEX，（6-8） 和生产带有非经典氨基酸的治疗性蛋白质。（9−12） 然而，发现新的非自然碱基始终是一项具有挑战性的任务，这些碱基具有来自天然碱基的外来结构支架，但具有理想的化学和生物特性。从 1960 年代开始，研究人员通过不同的氢键模式、疏水堆积相互作用和形状互补性等变体设计策略探索了数百个非天然碱基对，（13−15） 代表性地产生了三种类型的碱基对，TPT3-NaM、（16,17） Ds-Px、（18） 和 P-Z。（19,20） 这些对具有高效率、保真度和生物正交功能。特别是，TPT3-NaM 最近可用于半合成生物体 （SSO） 中，以完成增加的遗传信息的存储和检索。（9,12,17） 尽管 TPT3-NaM 的发现代表了非天然碱基对在合成生物学中应用的里程碑，但鉴于其进一步的应用，也发现了这对碱基对的一些缺点。TPT3-NaM 对的光敏性已被指出是对细胞健康生长的潜在威胁。（21−23） 通常，TPT3 的强烈近可见光吸收会导致细胞培养物中 ROS 的急剧增加，从而导致细胞的不可逆破坏。 据我们所知，提高非天然碱基对的光稳定性的方法仍然搁浅。
开发非天然碱基对 （UBP） 以扩展遗传字母表为增加 DNA 的信息存储容量提供了机会，（1-3） 同时为有前途的生物技术应用提供了一个平台，包括核酸的位点特异性修饰，（4,5） 高亲和力适配体的 SELEX，（6-8） 和生产带有非经典氨基酸的治疗性蛋白质。（9−12） 然而，发现新的非自然碱基始终是一项具有挑战性的任务，这些碱基具有来自天然碱基的外来结构支架，但具有理想的化学和生物特性。从 1960 年代开始，研究人员通过不同的氢键模式、疏水堆积相互作用和形状互补性等变体设计策略探索了数百个非天然碱基对，（13−15） 代表性地产生了三种类型的碱基对，TPT3-NaM、（16,17） Ds-Px、（18） 和 P-Z。（19,20） 这些对具有高效率、保真度和生物正交功能。特别是，TPT3-NaM 最近可用于半合成生物体 （SSO） 中，以完成增加的遗传信息的存储和检索。（9,12,17） 尽管 TPT3-NaM 的发现代表了非天然碱基对在合成生物学中应用的里程碑，但鉴于其进一步的应用，也发现了这对碱基对的一些缺点。TPT3-NaM 对的光敏性已被指出是对细胞健康生长的潜在威胁。（21−23） 通常，TPT3 的强烈近可见光吸收会导致细胞培养物中 ROS 的急剧增加，从而导致细胞的不可逆破坏。 据我们所知，提高非天然碱基对的光稳定性的方法仍然搁浅。
The efficiency and fidelity of unnatural base pairs play a dominant role in their replication, transcription, and translation procedures in vitro and in vivo. A process for producing the new structural scaffolds of unnatural base pairs usually comprises three steps: (1) chemical synthesis of nucleotides, (2) screening their efficiency and fidelity, (3) structure–activity relationship (SAR) feedback, and redesign. Using such a synthesis-screening strategy, a series of described base pairs are discovered, and valuable structure features for optimal unnatural base pairs can be concluded. (3,24,25) Nevertheless, the current methods omitted the evaluations of photostability of unnatural base pairs; it is even unclear about which parts of the optimized unnatural base pairs can be ascribed to their instabilities under light irradiation conditions. In the case of the TPT3 base, SAR investigations have shown that the C═S group is essential to keep the functions of TPT3 for efficient replications, (26,27) but the C═S groups are traditionally considered as photosensitive motifs. (28−30) In this regard, it will be quite difficult to enhance the photostability of TPT3-type nucleotides and, at the same time, keep their high efficiency and fidelity.
非天然碱基对的效率和保真度在其体外和体内的复制、转录和翻译过程中起着主导作用。产生非天然碱基对的新结构支架的过程通常包括三个步骤：（1） 核苷酸的化学合成，（2） 筛选它们的效率和保真度，（3） 构效关系 （SAR） 反馈和重新设计。使用这种合成筛选策略，发现了一系列描述的碱基对，并且可以得出最佳非自然碱基对的有价值的结构特征。（3,24,25） 然而，目前的方法省略了对非天然碱基对的光稳定性的评估;甚至不清楚优化的非天然碱基对的哪些部分可以归因于它们在光照射条件下的不稳定性。在 TPT3 碱基的情况下，SAR 研究表明，C═S 基团对于保持 TPT3 的功能以进行有效复制至关重要 （26,27），但 C═S 基团传统上被认为是光敏基序。（28−30） 在这方面，要提高 TPT3 型核苷酸的光稳定性，同时保持其高效率和保真度将是相当困难的。
非天然碱基对的效率和保真度在其体外和体内的复制、转录和翻译过程中起着主导作用。产生非天然碱基对的新结构支架的过程通常包括三个步骤：（1） 核苷酸的化学合成，（2） 筛选它们的效率和保真度，（3） 构效关系 （SAR） 反馈和重新设计。使用这种合成筛选策略，发现了一系列描述的碱基对，并且可以得出最佳非自然碱基对的有价值的结构特征。（3,24,25） 然而，目前的方法省略了对非天然碱基对的光稳定性的评估;甚至不清楚优化的非天然碱基对的哪些部分可以归因于它们在光照射条件下的不稳定性。在 TPT3 碱基的情况下，SAR 研究表明，C═S 基团对于保持 TPT3 的功能以进行有效复制至关重要 （26,27），但 C═S 基团传统上被认为是光敏基序。（28−30） 在这方面，要提高 TPT3 型核苷酸的光稳定性，同时保持其高效率和保真度将是相当困难的。
In this paper, a panel of unnatural bases was designed and synthesized, which had the basic structural skeletons of TPT3 but allowed the modifications of thiophene rings for optimizing their optical properties (Figure 1A). By testing their photostability in near-visible light to visible light, the more photostable analogues of dTPT3 were unveiled. Importantly, the following kinetic experiments and PCR-sequencing assay showed that one of the pairs (dNaM-dTAT1) had almost equally high efficiency and fidelity with the dNaM-dTPT3 pair itself. dNaM-dTAT1 could also be used effectively in E. coli cells for in vivo replications, and the cells cultured with dNaMTP and dTAT1TP showed more robust growth with less generation of reactive oxygen species (ROS). Our experiments not only demonstrate that the enhancements of photostability of TPT3-type unnatural base can be realized by local structural modifications for the first time but also result in a new dTAT1TP with high replication efficacy and stability, which can be used as a reformative tool in both of in vitro and in vivo assays for genetic code extensions.
在本文中，设计并合成了一组非天然碱基，它具有 TPT3 的基本结构骨架，但允许修改噻吩环以优化其光学特性（图 1A）。通过在近可见光到可见光下测试它们的光稳定性，揭示了 dTPT3 的更多光稳定性类似物。重要的是，以下动力学实验和 PCR 测序分析表明，其中一对 （dNaM-dTAT1） 与 dNaM-dTPT3 对本身具有几乎相同的效率和保真度。dNaM-dTAT1 也可以有效地用于大肠杆菌细胞进行体内复制，并且用 dNaMTP 和 dTAT1TP 培养的细胞显示出更稳健的生长和更少的活性氧 （ROS） 产生。我们的实验不仅表明，TPT3 型非天然碱基的光稳定性增强首次可以通过局部结构修饰来实现，而且还产生了具有高复制效率和稳定性的新型 dTAT1TP，可用作遗传密码延伸的体外和体内检测的改良工具。
在本文中，设计并合成了一组非天然碱基，它具有 TPT3 的基本结构骨架，但允许修改噻吩环以优化其光学特性（图 1A）。通过在近可见光到可见光下测试它们的光稳定性，揭示了 dTPT3 的更多光稳定性类似物。重要的是，以下动力学实验和 PCR 测序分析表明，其中一对 （dNaM-dTAT1） 与 dNaM-dTPT3 对本身具有几乎相同的效率和保真度。dNaM-dTAT1 也可以有效地用于大肠杆菌细胞进行体内复制，并且用 dNaMTP 和 dTAT1TP 培养的细胞显示出更稳健的生长和更少的活性氧 （ROS） 产生。我们的实验不仅表明，TPT3 型非天然碱基的光稳定性增强首次可以通过局部结构修饰来实现，而且还产生了具有高复制效率和稳定性的新型 dTAT1TP，可用作遗传密码延伸的体外和体内检测的改良工具。
Figure 1 图 1
Figure 1. (A) Design and structures of TPT3-type unnatural base nucleosides. (B) UV–vis spectra of the synthesized TPT3-type unnatural base nucleosides.
图 1.（A） TPT3 型非天然碱基核苷的设计和结构。（B） 合成的 TPT3 型非天然碱基核苷的紫外-可见光谱。
Results and Discussion 结果与讨论
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Design and Synthesis of TPT3-Type Unnatural Base Nucleotides
TPT3 型非天然碱基核苷酸的设计与合成
Based on data analysis of the kinetics and structural studies, an induced-fit mechanism is suggested for explaining the replications of hydrophobic unnatural base pairs, e.g., 5SICS-MMO2, 5SICS-NaM, TPT3-NaM, and PTMO-TPT3. (25,31−33) In the model, the pairing of unnatural bases in the polymerase active site drives the same large conformational change of the polymerase that is caused by the pairing of natural bases. During this procedure, the formation of the DNA-polymerase complex induces the unnatural bases to adopt an edge-to-edge paired Watson–Crick-like structure. However, a cross-strand intercalated structure forms in the free duplex DNA after an unnatural base pair synthesis and polymerase translocation. In order to continue the DNA synthesis, a de-intercalation occurs to reshape the Watson–Crick-like structure from the cross-strand intercalated structure. The interaction of each unnatural base is key for their efficient switches from intercalation to de-intercalation, which can also be designable in comprehensive consideration of SAR data including size-complementarity, π-stacking, and hydrophobic and hydrophilic forces.
基于动力学和结构研究的数据分析，提出了一种诱导拟合机制来解释疏水性非自然碱基对的复制，例如 5SICS-MMO2、5SICS-NaM、TPT3-NaM 和 PTMO-TPT3。（25,31−33） 在该模型中，聚合酶活性位点中非天然碱基的配对驱动了聚合酶的较大构象变化，这与天然碱基配对引起的相同。在此过程中，DNA-聚合酶复合物的形成诱导非天然碱基采用边对边配对的 Watson-Crick 样结构。然而，在非自然碱基对合成和聚合酶易位后，游离双链 DNA 中会形成交叉链嵌入结构。为了继续 DNA 合成，发生脱嵌以从交叉链插层结构重塑 Watson-Crick 样结构。每个非天然碱基的相互作用是它们从嵌入到脱嵌有效转换的关键，这也可以在综合考虑 SAR 数据（包括尺寸互补、π堆叠以及疏水和亲水力）的情况下进行设计。
基于动力学和结构研究的数据分析，提出了一种诱导拟合机制来解释疏水性非自然碱基对的复制，例如 5SICS-MMO2、5SICS-NaM、TPT3-NaM 和 PTMO-TPT3。（25,31−33） 在该模型中，聚合酶活性位点中非天然碱基的配对驱动了聚合酶的较大构象变化，这与天然碱基配对引起的相同。在此过程中，DNA-聚合酶复合物的形成诱导非天然碱基采用边对边配对的 Watson-Crick 样结构。然而，在非自然碱基对合成和聚合酶易位后，游离双链 DNA 中会形成交叉链嵌入结构。为了继续 DNA 合成，发生脱嵌以从交叉链插层结构重塑 Watson-Crick 样结构。每个非天然碱基的相互作用是它们从嵌入到脱嵌有效转换的关键，这也可以在综合考虑 SAR 数据（包括尺寸互补、π堆叠以及疏水和亲水力）的情况下进行设计。
TPT3 is discovered from its precursor 5SICS based on the induced-fit mechanism. (24) The size-complementarity of 5SICS and NaM is obtained by optimizing and screening from an over 3600 number of hydrophobic unnatural base pair candidates. (34) A fine adjustment on the benzene ring of 5SICS to reduce the hydrophobic surface of the base yields TPT3 with higher incorporation and extension efficiency. (24) A possible explanation is that the more polarizable TPT3 base is prone to drive the de-intercalation to form a Watson–Crick-like structure for efficient replications. On the other hand, the C═S groups in 5SICS and TPT3 are discovered to play critical roles for minor groove interactions. (26) Changing the C═S group to C═O group can cause a sharp reduction of its replication efficiency. (34) Therefore, keeping the geometric shape of TPT3 and the key C═S group, it is still possible to obtain new unnatural bases with better performances through careful optimizations of the interactions of TPT3 and its partner pairs. At this point, we designed a panel of TPT3-type unnatural bases as shown in Figure 1A. The nucleobases of dTPT4, dTAT1, and d4TFP (35) were synthesized from 2-thenaldehyde, thiazole-4-carboxaldehyde, and furfural via an intermolecular Curtius rearrangement based on literature methods, (36,37) and then the bases were coupled to (2R,5R)-5-chloro-2-(((4-methylbenzoyl)oxy)-methyl) tetrahydrofuran-3-yl 4-methylbenzoate. Column chromatography was used to obtain the β-anomer of dTPT4 and d4TFP as well as an α/β-anomer mix of dTAT1. The β-anomer of dTAT1 was obtained by reversed-phase HPLC. Then, they were converted to triphosphates under Ludwig conditions and purified by a Sephadex chromatography column and HPLC. The modifications on the thiophene ring of TPT3 via inducing the heteroatom do not affect the primary shapes of this base; however, it changes the UV–vis absorption property due to the variant conjugated systems (Figure 1B). Nucleosides d4TFP and dTPT4 show shorter absorption wavelengths, and dTAT1 shows a small molar absorbance coefficient in near-visible light spectra in contrast to dTPT3, thus offering potential candidates for the discovery of less photosensitive unnatural bases.
TPT3 是基于诱导拟合机制从其前体 5SICS 中发现的。（24） 5SICS 和 NaM 的大小互补性是通过从 3600 多个疏水性非天然碱基对候选者中进行优化和筛选而获得的。（34） 对 5SICS 的苯环进行微调以减少碱的疏水表面，得到具有更高掺入和延伸效率的 TPT3。（24） 一种可能的解释是，更易极化的 TPT3 碱基容易驱动脱嵌形成 Watson-Crick 样结构，以实现有效复制。另一方面，发现 5SICS 和 TPT3 中的 C═S 基团在小沟相互作用中起关键作用。（26） 将 C═S 组更改为 C═O 组会导致其复制效率急剧降低。（34） 因此，在保持 TPT3 的几何形状和关键的 C═S 基团的情况下，通过仔细优化 TPT3 及其伙伴对的相互作用，仍然有可能获得具有更好性能的新非天然碱基。此时，我们设计了一组 TPT3 型非天然碱基，如图 1A 所示。dTPT4、dTAT1 和 d4TFP 的核碱基 （35） 由2-乙醛、噻唑-4-甲醛和糠醛通过分子间 Curtius 重排合成 （36,37），然后将碱基偶联到 （2R，5 R）-5-氯-2-（（（4-甲基苯甲酰基）氧）-甲基）四氢呋喃-3-基 4-甲基苯甲酸酯上。柱层析用于获得 dTPT4 和 d4TFP 的 β 异头异构体以及 dTAT1 的 α/β 异头异构体混合物。dTAT1 的 β 异构体是通过反相 HPLC 获得的。 然后，在 Ludwig 条件下将它们转化为三磷酸盐，并通过 Sephadex 色谱柱和 HPLC 纯化。通过诱导杂原子对 TPT3 的噻吩环的修饰不会影响该碱基的初级形状;但是，由于变体共轭系统，它改变了 UV-Vis 吸收特性（图 1B）。核苷 d4TFP 和 dTPT4 显示出较短的吸收波长，与 dTPT3 相比，dTAT1 在近可见光光谱中显示出较小的摩尔吸光度系数，因此为发现光敏性较低的非天然碱基提供了潜在的候选者。
TPT3 是基于诱导拟合机制从其前体 5SICS 中发现的。（24） 5SICS 和 NaM 的大小互补性是通过从 3600 多个疏水性非天然碱基对候选者中进行优化和筛选而获得的。（34） 对 5SICS 的苯环进行微调以减少碱的疏水表面，得到具有更高掺入和延伸效率的 TPT3。（24） 一种可能的解释是，更易极化的 TPT3 碱基容易驱动脱嵌形成 Watson-Crick 样结构，以实现有效复制。另一方面，发现 5SICS 和 TPT3 中的 C═S 基团在小沟相互作用中起关键作用。（26） 将 C═S 组更改为 C═O 组会导致其复制效率急剧降低。（34） 因此，在保持 TPT3 的几何形状和关键的 C═S 基团的情况下，通过仔细优化 TPT3 及其伙伴对的相互作用，仍然有可能获得具有更好性能的新非天然碱基。此时，我们设计了一组 TPT3 型非天然碱基，如图 1A 所示。dTPT4、dTAT1 和 d4TFP 的核碱基 （35） 由2-乙醛、噻唑-4-甲醛和糠醛通过分子间 Curtius 重排合成 （36,37），然后将碱基偶联到 （2R，5 R）-5-氯-2-（（（4-甲基苯甲酰基）氧）-甲基）四氢呋喃-3-基 4-甲基苯甲酸酯上。柱层析用于获得 dTPT4 和 d4TFP 的 β 异头异构体以及 dTAT1 的 α/β 异头异构体混合物。dTAT1 的 β 异构体是通过反相 HPLC 获得的。 然后，在 Ludwig 条件下将它们转化为三磷酸盐，并通过 Sephadex 色谱柱和 HPLC 纯化。通过诱导杂原子对 TPT3 的噻吩环的修饰不会影响该碱基的初级形状;但是，由于变体共轭系统，它改变了 UV-Vis 吸收特性（图 1B）。核苷 d4TFP 和 dTPT4 显示出较短的吸收波长，与 dTPT3 相比，dTAT1 在近可见光光谱中显示出较小的摩尔吸光度系数，因此为发现光敏性较低的非天然碱基提供了潜在的候选者。
Photostability Evaluations of dTPT3, dTPT4, dTAT1, and d4TFP
dTPT3、dTPT4、dTAT1 和 d4TFP 的光稳定性评估
Visible light and near-visible light are highly abundant in natural and standard fluorescence experimental environments. (38) The TPT3-type unnatural base nucleosides, dTPT3, dTPT4, dTAT1, and d4TFP, showed strong absorption within the near-visible light spectrum (350–410 nm) using UV–vis spectra (Figure 1B). To investigate the photostability of these nucleosides, they were dissolved in phosphate-buffered saline (pH = 7.4) and exposed to near-visible light and visible light (red, green, blue, and purple) for 0.25, 0.5, 0.75, 1, and 1.25 h. Then, samples were analyzed by HPLC and the degradation of nucleosides was normalized with samples without exposure (Figure 2A–D, Figure S1). The results showed that both TPT3 and its analogues were stable to visible light. When exposed to near-visible light, dTPT3, dTPT4, and dTAT1 were degraded continuously with the prolonged time, and the degradation of d4TFP was terminated at 0.5 h. In addition, the percent remaining of dTPT3, dTPT4, dTAT1, and d4TFP were 0.28 ± 0.02, 0.47 ± 0.03, 0.73 ± 0.01, and 0.85 ± 0.02 at 1.25 h, respectively. These data indicate that the nucleobases dTAT1 and d4TFP are more photostable. The changes in the chemical structure on the thiophene motif of TPT3 did change the photostability of these unnatural base nucleosides. Thus, the propriate modifications on the thiophene motif of TPT3 can offer a way to improve the photostability of TPT3-type bases.
可见光和近可见光在自然和标准荧光实验环境中非常丰富。（38） 使用紫外-可见光谱，TPT3 型非天然碱基核苷 dTPT3、dTPT4、dTAT1 和 d4TFP 在近可见光谱 （350-410 nm） 内显示出强吸收（图 1B）。为了研究这些核苷的光稳定性，将它们溶解在磷酸盐缓冲盐水 （pH = 7.4） 中，并暴露在近可见光和可见光（红色、绿色、蓝色和紫色）下 0.25、0.5、0.75、1 和 1.25 小时。然后，通过 HPLC 分析样品，并用未暴露的样品对核苷的降解进行归一化（图 2A-D，图 S1）。结果表明，TPT3 及其类似物对可见光均稳定。当暴露在近可见光下时，dTPT3 、 dTPT4 和 dTAT1 随着时间的延长而不断降解，d4TFP 的降解在 0.5 h 终止。此外，dTPT3 、 dTPT4 、 dTAT1 和 d4TFP 的剩余百分比分别为 0.28 ± 0.02 、 0.47 ± 0.03 、 0.73 ± 0.01 和 0.85 ± 0.02。这些数据表明，核碱基 dTAT1 和 d4TFP 的光稳定性更强。TPT3 噻吩基序上化学结构的变化确实改变了这些非天然碱基核苷的光稳定性。因此，对 TPT3 噻吩基序的适当修饰可以提供一种提高 TPT3 型碱基光稳定性的方法。
可见光和近可见光在自然和标准荧光实验环境中非常丰富。（38） 使用紫外-可见光谱，TPT3 型非天然碱基核苷 dTPT3、dTPT4、dTAT1 和 d4TFP 在近可见光谱 （350-410 nm） 内显示出强吸收（图 1B）。为了研究这些核苷的光稳定性，将它们溶解在磷酸盐缓冲盐水 （pH = 7.4） 中，并暴露在近可见光和可见光（红色、绿色、蓝色和紫色）下 0.25、0.5、0.75、1 和 1.25 小时。然后，通过 HPLC 分析样品，并用未暴露的样品对核苷的降解进行归一化（图 2A-D，图 S1）。结果表明，TPT3 及其类似物对可见光均稳定。当暴露在近可见光下时，dTPT3 、 dTPT4 和 dTAT1 随着时间的延长而不断降解，d4TFP 的降解在 0.5 h 终止。此外，dTPT3 、 dTPT4 、 dTAT1 和 d4TFP 的剩余百分比分别为 0.28 ± 0.02 、 0.47 ± 0.03 、 0.73 ± 0.01 和 0.85 ± 0.02。这些数据表明，核碱基 dTAT1 和 d4TFP 的光稳定性更强。TPT3 噻吩基序上化学结构的变化确实改变了这些非天然碱基核苷的光稳定性。因此，对 TPT3 噻吩基序的适当修饰可以提供一种提高 TPT3 型碱基光稳定性的方法。
Figure 2 图 2
Figure 2. Photostability of dTPT3 and its analogues. (A) Photostability of dTPT3; (B) photostability of dTPT4; (C) photostability of dTAT1; (D) photostability of d4TFP.
图 2.dTPT3 及其类似物的光稳定性。（A） dTPT3 的光稳定性;（B） dTPT4 的光稳定性;（C） dTAT1 的光稳定性;（D） d4TFP 的光稳定性。
In Vitro Efficiency and Fidelity Evaluations of dTPT4TP, dTAT1TP, and d4TFPTP
dTPT4TP、dTAT1TP 和 d4TFPTP 的体外效率和保真度评估
The incorporation and extension of dTPT3 analogues were analyzed by presteady-state kinetics. (16,25,39) The assay was performed by extending a 23-mer primer labeled with HEX fluorescence (green) opposite its cognate 45-mer template using the Klenow fragment of E. coli DNA polymerase I (Kf) (Figure 3A). The incorporation was characterized by measuring the extending ratio of the unnatural base and next correct triphosphate (dCTP), demonstrating as ≥24-mer/all, and the extension was characterized by measuring the extending ratio of the next correct triphosphate (dCTP), demonstrated as ≥25-mer/≥24-mer. The separate extension assay was also performed by adding dYPT for a preincubation to allow for full incorporation and then adding dCTP. Full-length analysis was also performed by adding the unnatural and four natural bases to explore whether the unnatural base pair on DNA would affect the chain extension. Under the conditions selected, the % incorporation of dTPT3TP, dTPT4TP, dTAT1TP, and d4TFPTP were 63.4, 61.7, 60.3, and 63.3, and the % extension ratio of dTPT3TP, dTPT4TP, dTAT1TP, and d4TFPTP were 97, 96.6, 95.5, and 96.4%, respectively (Table 1). The % separate extension ratio of dTPT3TP, dTPT4TP, dTAT1TP, and d4TFPTP were 94.7 ± 0.5, 94.9 ± 0.5, 94.5 ± 0.2, and 95.5 ± 0.3, respectively (Figure S2 and Table S1). The analogues showed a narrow range of behaviors. As reported, dTPT3 can be weakly paired with natural nucleotides depending on the sequence context. (39) It was found that dTPT3 was mispaired with dGTP in the incorporation assay or dCTP in the extension assay (Table 1 and Figure 3B,C). However, the mispaired oligonucleotides could not be further extended (Figure S3). The other analogues showed less mispairing with dGTP or dCTP than dTPT3, especially the d4TFP showed barely mispairing. Full-length fragments with dTPT3 and its analogues were obtained and showed no significant difference, indicating that modifications of dTPT3 analogues did not affect the ability of DNA chain extension (Figure S4).
通过预稳态动力学分析 dTPT3 类似物的掺入和延伸。（16,25,39） 通过使用大肠杆菌 DNA 聚合酶 I （Kf） 的 Klenow 片段，将用 HEX 荧光（绿色）标记的 23 聚体引物延伸到其同源 45 聚体模板的对面进行测定（图 3A）。通过测量非天然碱基和下一个正确三磷酸盐 （dCTP） 的延伸比来表征掺入，证明为 ≥24-mer/all，通过测量下一个正确三磷酸盐 （dCTP） 的延伸比来表征延伸，证明为 ≥25-mer/≥24-mer。还通过添加 dYPT 进行预孵育以允许完全掺入，然后添加 dCTP 进行单独的延伸测定。还通过添加非天然碱基和 4 个天然碱基进行全长分析，以探讨 DNA 上的非天然碱基对是否会影响链延伸。在所选条件下，dTPT3TP、dTPT4TP、dTAT1TP 和 d4TFPTP 的掺入百分比分别为 63.4、61.7、60.3 和 63.3，dTPT3TP、dTPT4TP、dTAT1TP 和 d4TFPTP 的百分比延伸率分别为 97、96.6、95.5 和 96.4%（表 1）。dTPT3TP、dTPT4TP、dTAT1TP 和 d4TFPTP 的 % 分离延伸比分别为 94.7 ± 0.5、94.9 ± 0.5、94.5 ± 0.2 和 95.5 ± 0.3（图 S2 和表 S1）。类似物表现出狭窄的行为范围。据报道，dTPT3 可以与天然核苷酸弱配对，具体取决于序列环境。（39） 发现 dTPT3 在掺入试验中与 dGTP 或延伸试验中的 dCTP 错配（表 1 和图 3B、C）。 然而，错配对的寡核苷酸无法进一步延伸（图 S3）。与 dTPT3 相比，其他类似物与 dGTP 或 dCTP 的错配更少，尤其是 d4TFP 几乎没有错配。获得带有 dTPT3 及其类似物的全长片段，显示无显着差异，表明 dTPT3 类似物的修饰不会影响 DNA 链延伸的能力（图 S4）。
通过预稳态动力学分析 dTPT3 类似物的掺入和延伸。（16,25,39） 通过使用大肠杆菌 DNA 聚合酶 I （Kf） 的 Klenow 片段，将用 HEX 荧光（绿色）标记的 23 聚体引物延伸到其同源 45 聚体模板的对面进行测定（图 3A）。通过测量非天然碱基和下一个正确三磷酸盐 （dCTP） 的延伸比来表征掺入，证明为 ≥24-mer/all，通过测量下一个正确三磷酸盐 （dCTP） 的延伸比来表征延伸，证明为 ≥25-mer/≥24-mer。还通过添加 dYPT 进行预孵育以允许完全掺入，然后添加 dCTP 进行单独的延伸测定。还通过添加非天然碱基和 4 个天然碱基进行全长分析，以探讨 DNA 上的非天然碱基对是否会影响链延伸。在所选条件下，dTPT3TP、dTPT4TP、dTAT1TP 和 d4TFPTP 的掺入百分比分别为 63.4、61.7、60.3 和 63.3，dTPT3TP、dTPT4TP、dTAT1TP 和 d4TFPTP 的百分比延伸率分别为 97、96.6、95.5 和 96.4%（表 1）。dTPT3TP、dTPT4TP、dTAT1TP 和 d4TFPTP 的 % 分离延伸比分别为 94.7 ± 0.5、94.9 ± 0.5、94.5 ± 0.2 和 95.5 ± 0.3（图 S2 和表 S1）。类似物表现出狭窄的行为范围。据报道，dTPT3 可以与天然核苷酸弱配对，具体取决于序列环境。（39） 发现 dTPT3 在掺入试验中与 dGTP 或延伸试验中的 dCTP 错配（表 1 和图 3B、C）。 然而，错配对的寡核苷酸无法进一步延伸（图 S3）。与 dTPT3 相比，其他类似物与 dGTP 或 dCTP 的错配更少，尤其是 d4TFP 几乎没有错配。获得带有 dTPT3 及其类似物的全长片段，显示无显着差异，表明 dTPT3 类似物的修饰不会影响 DNA 链延伸的能力（图 S4）。
Figure 3 图 3
Figure 3. Kinetics analysis and sequencing analysis of the PCR products. (A) Kf-mediated primer extension and sequences used in this study; (B) representative gel for the incorporation assay; (C) representative gel for the extension assay. (D) Sequencing analysis of the PCR products after 36 cycles by OneTaq polymerase.
图 3.PCR 产物的动力学分析和测序分析。（A） 本研究中使用的 Kf 介导的引物延伸和序列;（B） 用于掺入测定的代表性凝胶;（C） 用于延伸测定的代表性凝胶。（D） 使用 OneTaq 聚合酶对 36 个循环后的 PCR 产物进行测序分析。
| dYTP | incorporation (%) 公司注册 （%） | mispairing (%) 错配 （%） | extension (%) 扩展 （%） | mispairing (%) 错配 （%） |
|---|---|---|---|---|
| dTPT3 | 63.4 ± 1.1 | 19.9 ± 3.8 | 97 ± 0.2 | 20.9 ± 1.1 |
| dTPT4 | 61.7 ± 1.2 | 1.4 ± 0.7 | 96.6 ± 0.2 | 7 ± 3.3 |
| dTAT1 | 60.3 ± 1.5 60,3 ± 1.5 | 2.7 ± 0.7 | 95.5 ± 0.3 | 11.2 ± 0.2 |
| d4TFP | 63.3 ± 2.0 | 1.6 ± 0.3 | 96.4 ± 0.7 | 3.0 ± 1.0 |
a
a
Incorporation assay conditions: 12.5 μM dYTP, 10 s. Extension assay conditions: 12.5 μM dYTP and dCTP, 10 s. The mispairing was characterized by measuring the paired ratio of the additional extension, demonstrated as 25-mer/≥24-mer or 26-mer/≥25-mer.
a
掺入测定条件：12.5 μM dYTP，10 秒。延伸测定条件：12.5 μM dYTP 和 dCTP，10 秒。通过测量额外延伸的配对比率来表征错配，显示为 25 mer/≥24 mer 或 26 mer/≥25-mer。
To further evaluate replication in vitro, a 134 bp single-stranded DNA containing dNaM (16) was synthesized. Due to the different sources and evolutionary levels, the differentiated and proofreading ability of polymerases is uneven, (40) and the adaptability of novel unnatural base pairs to polymerases should also be screened. (41,42) The template was amplified by PCR with HieffTaq, OneTaq, and Taq polymerase. Different polymerases were used for the amplification of 36 cycles to explore the enzyme adaptability of the nucleobases. The unnatural base pair retention per doubling defined as fidelity was calculated from the percentage of the amplified DNA that retained the unnatural base pair (retention). With HieffTaq, the fidelity of DNA containing dNaM-dTPT3, dNaM-dTAT1, dNaM-dTPT4, and dNaM-d4TFP, was 99.8, 99.8, 99.7, and 99.4% (Table 2, and Figure S5). With OneTaq, the fidelity of DNA containing dNaM-dTPT3, dNaM-dTAT1, dNaM-dTPT4, and dNaM-d4TFP was 99.5, 99.5, 99, and 99% (Table 2, and Figure S6). With Taq, the fidelity of DNA containing dNaM-dTPT3, dNaM-dTAT1, dNaM-dTPT4, and dNaM-d4TFP, was 99.8, 99.9, 99.6, and 98.2% (Table 2 and Figure S7). With the three polymerases, DNA containing dNaM-dTPT3 and dNaM-dTAT1 was amplified with essentially the same retention and fidelity; however, DNA containing dNaM-dTPT4, and especially dNaM-d4TFP, was amplified with significantly lower retention and fidelity. With OneTaq, the retention and fidelity of DNA containing the four unnatural base pairs were significantly lower than with HieffTaq and Taq polymerases, which may be due to the differentiated and proofreading ability of polymerases, as OneTaq has the proofreading ability and the other two do not have. These suggest that the optimization of UBPs should take into account the influence of different DNA polymerases. In summary, the dTPT3 and its analogues are adaptive to different enzymes and dTAT1 performs similarly to dTPT3 in PCR.
为了进一步评估体外复制，合成了含有 dNaM （16） 的 134 bp 单链 DNA。由于来源和进化水平不同，聚合酶的分化和校对能力不均衡 （40），还应筛选新的非天然碱基对聚合酶的适应性。（41,42） 使用 HieffTaq、OneTaq 和 Taq 聚合酶通过 PCR 扩增模板。使用不同的聚合酶进行 36 个循环的扩增，以探索核碱基的酶适应性。定义为保真度的每次倍增的非自然碱基对保留率是根据保留非天然碱基对的扩增 DNA 的百分比（保留）计算得出的。使用 HieffTaq，含有 dNaM-dTPT3、dNaM-dTAT1、dNaM-dTPT4 和 dNaM-d4TFP 的 DNA 的保真度分别为 99.8%、99.8%、99.7% 和 99.4%（表 2 和图 S5）。使用 OneTaq 时，含有 dNaM-dTPT3、dNaM-dTAT1、dNaM-dTPT4 和 dNaM-d4TFP 的 DNA 的保真度分别为 99.5%、99.5%、99% 和 99%（表 2 和图 S6）。使用 Taq 时，含有 dNaM-dTPT3、dNaM-dTAT1、dNaM-dTPT4 和 dNaM-d4TFP 的 DNA 的保真度分别为 99.8%、99.9%、99.6% 和 98.2%（表 2 和图 S7）。使用三种聚合酶，含有 dNaM-dTPT3 和 dNaM-dTAT1 的 DNA 以基本相同的保留和保真度扩增;然而，含有 dNaM-dTPT4 的 DNA，尤其是 dNaM-d4TFP 的扩增保留率和保真度显著降低。 使用 OneTaq 时，含有四个非天然碱基对的 DNA 的保留率和保真度显著低于 HieffTaq 和 Taq 聚合酶，这可能是由于聚合酶的分化和校对能力，因为 OneTaq 具有校对能力，而其他两种没有。这些表明 UBP 的优化应考虑不同 DNA 聚合酶的影响。总之，dTPT3 及其类似物可适应不同的酶，并且 dTAT1 在 PCR 中的表现与 dTPT3 相似。
为了进一步评估体外复制，合成了含有 dNaM （16） 的 134 bp 单链 DNA。由于来源和进化水平不同，聚合酶的分化和校对能力不均衡 （40），还应筛选新的非天然碱基对聚合酶的适应性。（41,42） 使用 HieffTaq、OneTaq 和 Taq 聚合酶通过 PCR 扩增模板。使用不同的聚合酶进行 36 个循环的扩增，以探索核碱基的酶适应性。定义为保真度的每次倍增的非自然碱基对保留率是根据保留非天然碱基对的扩增 DNA 的百分比（保留）计算得出的。使用 HieffTaq，含有 dNaM-dTPT3、dNaM-dTAT1、dNaM-dTPT4 和 dNaM-d4TFP 的 DNA 的保真度分别为 99.8%、99.8%、99.7% 和 99.4%（表 2 和图 S5）。使用 OneTaq 时，含有 dNaM-dTPT3、dNaM-dTAT1、dNaM-dTPT4 和 dNaM-d4TFP 的 DNA 的保真度分别为 99.5%、99.5%、99% 和 99%（表 2 和图 S6）。使用 Taq 时，含有 dNaM-dTPT3、dNaM-dTAT1、dNaM-dTPT4 和 dNaM-d4TFP 的 DNA 的保真度分别为 99.8%、99.9%、99.6% 和 98.2%（表 2 和图 S7）。使用三种聚合酶，含有 dNaM-dTPT3 和 dNaM-dTAT1 的 DNA 以基本相同的保留和保真度扩增;然而，含有 dNaM-dTPT4 的 DNA，尤其是 dNaM-d4TFP 的扩增保留率和保真度显著降低。 使用 OneTaq 时，含有四个非天然碱基对的 DNA 的保留率和保真度显著低于 HieffTaq 和 Taq 聚合酶，这可能是由于聚合酶的分化和校对能力，因为 OneTaq 具有校对能力，而其他两种没有。这些表明 UBP 的优化应考虑不同 DNA 聚合酶的影响。总之，dTPT3 及其类似物可适应不同的酶，并且 dTAT1 在 PCR 中的表现与 dTPT3 相似。
| dTPT3 | dTPT4 | dTAT1 | d4TFP | |||||
|---|---|---|---|---|---|---|---|---|
| dXTP | retention (R, %) 留存率 （R， %） | fidelity (R, %) 保真度 （R， %） | retention (R, %) 留存率 （R， %） | fidelity (R, %) 保真度 （R， %） | retention (R, %) 留存率 （R， %） | fidelity (R, %) 保真度 （R， %） | retention (R, %) 留存率 （R， %） | fidelity (R, %) |
| Hieff 希夫 | 93.9 ± 1.7 93,9 ± 1.7 | 99.8 ± 0.04 | 88.6 ± 0.8 | 99.7 ± 0.02 | 94.4 ± 1.3 | 99.8 ± 0.05 | 79.7 ± 2.2 | 99.4 ± 0.08 |
| OneTaq OneTaq 公司 | 84.2 ± 1.2 | 99.5 ± 0.04c 99.5 ± 0.04摄氏度 | 70.2 ± 2.7 | 99 ± 0.1 | 84.4 ± 1.6 | 99.5 ± 0.05 | 68.9 ± 1.3 | 99 ± 0.05 |
| Taq | 93.6 ± 0.4 | 99.8 ± 0.01 | 85.2 ± 1 | 99.6 ± 0.03 | 95.5 ± 1.4 | 99.9 ± 0.04 | 52 ± 4.7 | 98.2 ± 0.2 |
| OneTaqb | 83.8 ± 0.3 | 99.7 ± 0.01 | 79 ± 1.6 | 99.5 ± 0.04 | ||||
a
a
The data indicate mean values ± SD. n = 3.
a
数据表示 SD ±平均值。n = 3。
b
b
Represents PCR that were amplified for 51 cycles, and others were amplificated for 36 cycles.
b
表示扩增 51 个循环的 PCR，其他 PCR 扩增 36 个循环。
c
c
Represented the significant difference of TPT3 in fidelity between continuous 36 cycles and then 3 x 17 cycles.
c
表示 TPT3 在连续 36 个循环和 3 x 17 个循环之间的保真度上的显着差异。
PCR, a thermal cycling reaction, is generally implemented to amplify DNA containing the unnatural base pair. (4) Unnatural base pairs with better thermostability will perform better in PCR. PCR with continuous and discontinuous cycles can be performed to evaluate their thermostability. To further characterize dTAT1, amplification of 51 cycles (3 × 17, PCR mix was amplified over 17 cycles, diluted to 1000-fold, and amplified over the second 17 cycles, and finally diluted 1000-fold and amplified over the third 17 cycles) with OneTaq polymerase was performed. The fidelities of dNaM-dTPT3 by 51 cycles were 99.7%, which was reduced by PCR of 36 continuous cycles (99.5%). Also, the fidelities of dNaM-dTAT1 by 51 and 36 cycles were both 99.5%, showing no differences (Table 2, Figures S6 and S8). The results suggest that dTPT3 is less thermostable. Amplified efficiency of DNA containing unnatural base pair represents the yield of PCR products; the limited amplified efficiency will lead to high cost and constraints in further research. Amplified efficiency was determined by monitoring the grayscale of the stripe employing 16 and 20 cycles PCR with OneTaq. Compared to dNaM-dTPT3, the amplified efficiency of dNaM-dTAT1 was about 98% (16 doublings: = 0.986; 20 doublings: = 0.983) (Figure S9). The results indicate that the dTAT1 has similar efficiency with dTPT3.
PCR 是一种热循环反应，通常用于扩增含有非天然碱基对的 DNA。（4） 具有更好热稳定性的非天然碱基对在 PCR 中表现更好。可以进行连续和不连续循环的 PCR 以评估其热稳定性。为了进一步表征 dTAT1，使用 OneTaq 聚合酶扩增 51 个循环（3 × 17 个循环，PCR 混合物在 17 个循环中扩增，稀释至 1000 倍，并在第二个 17 个循环中扩增，最后稀释 1000 倍并在第三个 17 个循环中扩增）。51 个循环的 dNaM-dTPT3 的保真度为 99.7%，通过 36 个连续循环的 PCR （99.5%） 降低。此外，dNaM-dTAT1 在 51 次和 36 次循环中的保真度均为 99.5%，没有差异（表 2，图 S6 和 S8）。结果表明 dTPT3 的热稳定性较差。含有非天然碱基对的 DNA 的扩增效率代表 PCR 产物的产量;有限的放大效率将导致高成本和进一步研究的限制。通过使用 OneTaq 进行 16 和 20 个循环 PCR 监测条带的灰度来确定扩增效率。与 dNaM-dTPT3 相比，dNaM-dTAT1 的扩增效率约为 98%（16 次倍增：= 0.986;20 次倍增：= 0.983）（图 S9）。结果表明，dTAT1 与 dTPT3 具有相似的效率。
PCR 是一种热循环反应，通常用于扩增含有非天然碱基对的 DNA。（4） 具有更好热稳定性的非天然碱基对在 PCR 中表现更好。可以进行连续和不连续循环的 PCR 以评估其热稳定性。为了进一步表征 dTAT1，使用 OneTaq 聚合酶扩增 51 个循环（3 × 17 个循环，PCR 混合物在 17 个循环中扩增，稀释至 1000 倍，并在第二个 17 个循环中扩增，最后稀释 1000 倍并在第三个 17 个循环中扩增）。51 个循环的 dNaM-dTPT3 的保真度为 99.7%，通过 36 个连续循环的 PCR （99.5%） 降低。此外，dNaM-dTAT1 在 51 次和 36 次循环中的保真度均为 99.5%，没有差异（表 2，图 S6 和 S8）。结果表明 dTPT3 的热稳定性较差。含有非天然碱基对的 DNA 的扩增效率代表 PCR 产物的产量;有限的放大效率将导致高成本和进一步研究的限制。通过使用 OneTaq 进行 16 和 20 个循环 PCR 监测条带的灰度来确定扩增效率。与 dNaM-dTPT3 相比，dNaM-dTAT1 的扩增效率约为 98%（16 次倍增：= 0.986;20 次倍增：= 0.983）（图 S9）。结果表明，dTAT1 与 dTPT3 具有相似的效率。
DFT Calculations for Model the Pairing of TPT3 Analogues with NaM
用于对 TPT3 类似物与 NaM 的配对进行建模的 DFT 计算
The above experimental findings indicated the effective formation of hydrophobic unnatural base pairs by all the TPT3 analogues and NaM. However, we also found that the fidelity of the pairs ranged slightly. It was noteworthy that the pair TAT1-NaM demonstrated similar fidelity with TPT3-NaM itself. Indeed, quantum mechanical calculations have been performed to explain the noncanonical base pairs that are formed through size-complementary, electrostatic, and hydrophobic interactions. (24,31−33,43) A Watson-Crick like geometry of TPT3-NaM pair is suggested by X-ray experiments. (44,45) In order to rationalize the differences observed when the TPT3 analogues paired with NaM, we carried out a DFT analysis as shown in Figure 4 and Table S1. The distances between the C1’ atoms of nucleotides in the optimized Watson-Crick like geometry of TPT3-NaM, TPT4-NaM, TAT1-NaM, and 4TFP-NaM are 10.72 Å, 10.77 Å, 10.00 Å, and 10.37 Å, respectively, which are very closed to the average value of A-T and G-C pairs (10.48 Å and 10.69 Å). In the case of TPT3-NaM and TAT1-NaM, the differences in their distances between the C1’ atoms of nucleotides were only 0.04 Å. Further, the interaction energy calculation showed less than 0.23 kcal/mol ranged from all the pairs (Table S2). The tiny differences in interaction energy of the four pairs might be ascribed to their different electrostatic potential (Figure 4). For example, replacing the aldehyde group of Pa with the nitro group changes the electrostatic potential (ESP) maps, thus preventing the mispairing with A. (46) Taken together, the above analysis indicated that our changes in the chemical structure on the thiophene motif of TPT3 did not cause dramatic differences in the geometries and energies of their pairing with NaM.
上述实验结果表明，所有 TPT3 类似物和 NaM 都能有效形成疏水性非天然碱基对。然而，我们还发现，货币对的保真度略有不同。值得注意的是，TAT1-NaM 对与 TPT3-NaM 本身表现出相似的保真度。事实上，已经进行了量子力学计算来解释通过尺寸互补、静电和疏水相互作用形成的非规范碱基对。（24,31−33,43）X 射线实验提出了 TPT3-NaM 对的 Watson-Crick 类似几何形状。（44,45） 为了合理化 TPT3 类似物与 NaM 配对时观察到的差异，我们进行了 DFT 分析，如图 4 和表 S1 所示。TPT3-NaM、TPT4-NaM、TAT1-NaM 和 4TFP-NaM 的优化 Watson-Crick 几何结构中核苷酸的 C1' 原子之间的距离分别为 10.72 Å、10.77 Å、10.00 Å 和 10.37 Å，这与 A-T 和 G-C 对的平均值（10.48 Å 和 10.69 Å）非常接近。在 TPT3-NaM 和 TAT1-NaM 的情况下，核苷酸的 C1' 原子之间的距离差异仅为 0.04 Å。此外，相互作用能计算显示所有对的含量范围小于 0.23 kcal/mol（表 S2）。四对相互作用能的微小差异可能归因于它们不同的静电电位（图 4）。例如，用硝基取代 Pa 的醛基会改变静电势 （ESP） 图，从而防止与 A 错配。 （46） 综上所述，上述分析表明，我们在 TPT3 的噻吩基序上化学结构的变化并没有导致它们与 NaM 配对的几何形状和能量发生显着差异。
上述实验结果表明，所有 TPT3 类似物和 NaM 都能有效形成疏水性非天然碱基对。然而，我们还发现，货币对的保真度略有不同。值得注意的是，TAT1-NaM 对与 TPT3-NaM 本身表现出相似的保真度。事实上，已经进行了量子力学计算来解释通过尺寸互补、静电和疏水相互作用形成的非规范碱基对。（24,31−33,43）X 射线实验提出了 TPT3-NaM 对的 Watson-Crick 类似几何形状。（44,45） 为了合理化 TPT3 类似物与 NaM 配对时观察到的差异，我们进行了 DFT 分析，如图 4 和表 S1 所示。TPT3-NaM、TPT4-NaM、TAT1-NaM 和 4TFP-NaM 的优化 Watson-Crick 几何结构中核苷酸的 C1' 原子之间的距离分别为 10.72 Å、10.77 Å、10.00 Å 和 10.37 Å，这与 A-T 和 G-C 对的平均值（10.48 Å 和 10.69 Å）非常接近。在 TPT3-NaM 和 TAT1-NaM 的情况下，核苷酸的 C1' 原子之间的距离差异仅为 0.04 Å。此外，相互作用能计算显示所有对的含量范围小于 0.23 kcal/mol（表 S2）。四对相互作用能的微小差异可能归因于它们不同的静电电位（图 4）。例如，用硝基取代 Pa 的醛基会改变静电势 （ESP） 图，从而防止与 A 错配。 （46） 综上所述，上述分析表明，我们在 TPT3 的噻吩基序上化学结构的变化并没有导致它们与 NaM 配对的几何形状和能量发生显着差异。
Figure 4 图 4
Figure 4. Optimized base pairs between dNaM and dTPT3 as well as its analogues (left). Optimal distances in the gas phase of the key atoms and C1′–C1′ distances. Oxygen, sulfur, and nitrogen are colored red, yellow, and blue, respectively. The electrostatic potential (ESP) maps (right) describe the electron density distribution; the red area represents the electron-rich area; and the green area represents the electron-deficient area.
图 4.dNaM 和 dTPT3 及其类似物之间的优化碱基对（左）。关键原子气相中的最佳距离和 C1′–C1′ 距离。氧、硫和氮分别呈红色、黄色和蓝色。静电势 （ESP） 图（右）描述了电子密度分布;红色区域代表电子富集区域;绿色区域代表缺电子区域。
Replications of dNaM-dTAT1 in Cells
dNaM-dTAT1 在细胞中的复制
Meanwhile, DNA polymerases in organisms are different from commercial DNA polymerases, so replication and transcription experiments in vivo are also necessary. (31,47) The effects of these nucleotides on physiology should be taken into account to build a healthier cellular system with unnatural nucleotides. (48) To evaluate replication in vivo, the previously reported D6 containing dNaM flanked on each side by three randomized natural nucleotides was synthesized. (16) The template was amplified by PCR using OneTaq polymerase in the presence of dNaM/dTPT3 and then inserted into plasmid pBLUE-T. The plasmid-expressed PtNTT2 to transport triphosphate was also synthesized. Then, the two plasmids were transformed to E. coli BL 21 (DE3) together. The workflow was shown (Figure 5A). After transformation, the E. coli BL 21 (DE3) was incubated at 37 °C for 17 h with dNaM/dTPT3 or dNaM/dTAT1 at a concentration of 125 μM. Finally, the retention was calculated by PCR using the E. coli cells as templates. It was found that the retentions of dNaM-dTAT1 and dNaM-dTPT3 in vivo were 51.4 ± 1.8 and 53.3 ± 4.4%, respectively. There were no differences between retentions of the two base pairs (Figure 5B and Figure S10). The retentions for in vivo replication are lower than reported previously, (17) which is likely due to the fact that a library of randomized sequences was employed. UBP retention is known to be sequence context-dependent with some sequence context showing high retention and others less or none. (48,49) The plasmids bearing randomized sequences will display the comprehensive retentions. In addition, the growth of E. coli cells was also analyzed by measuring OD600, and we found that E. coli cells incubated with dNaM/dTAT1 showed more robust growth (Figure 5C). The dTAT1 or dTPT3 was also individually incubated with E. coli cells. Also, the growth of E. coli cells showed the same trend (Figure 5D). As reported, UBPs incubated with E. coli cells can lead to the generation of reactive oxygen species (ROS). (23) The level of ROS was also monitored using the DCFH-DA probe, and we found that dTAT1 resulted in a lower ROS level (Figure 5E). These suggested that the new dTAT1 was less toxic to E. coli cells.
同时，生物体中的 DNA 聚合酶与市售的 DNA 聚合酶不同，因此还需要在体内进行复制和转录实验。（31,47） 应考虑这些核苷酸对生理学的影响，以使用非天然核苷酸构建更健康的细胞系统。（48） 为了评估体内复制，合成了先前报道的含有 dNaM 的 D6，每侧两侧有三个随机的天然核苷酸。（16） 在 dNaM/dTPT3 存在下，使用 OneTaq 聚合酶通过 PCR 扩增模板，然后插入质粒 pBLUE-T 中。还合成了表达质粒的 PtNTT2 以转运三磷酸盐。然后，将两个质粒一起转化到大肠杆菌 BL 21 （DE3） 中。显示了工作流程（图 5A）。转化后，将大肠杆菌 BL 21 （DE3） 与 125 μM 浓度的 dNaM/dTPT3 或 dNaM/dTAT1 在 37 °C 下孵育 17 小时。最后，以大肠杆菌细胞为模板，通过 PCR 计算保留率。结果发现 dNaM-dTAT1 和 dNaM-dTPT3 在体内的保留分别为 51.4 ± 1.8 和 53.3 ± 4.4%。两个碱基对的保留率没有差异（图 5B 和图 S10）。体内复制的保留率低于之前报道的 （17），这可能是由于采用了随机序列库。众所周知，UBP 保留率与序列上下文相关，某些序列上下文显示高保留率，而另一些则较低或没有保留率。（48,49） 带有随机序列的质粒将显示综合保留。此外，E 的生长。 还通过测量 OD600 分析了大肠杆菌细胞，我们发现与 dNaM/dTAT1 一起孵育的大肠杆菌细胞显示出更强劲的生长（图 5C）。dTAT1 或 dTPT3 也与大肠杆菌细胞单独孵育。此外，大肠杆菌细胞的生长也显示出相同的趋势（图 5D）。据报道，与大肠杆菌细胞一起孵育的 UBP 可导致活性氧 （ROS） 的产生。（23） 还使用 DCFH-DA 探针监测 ROS 水平，我们发现 dTAT1 导致 ROS 水平降低（图 5E）。这些表明新的 dTAT1 对大肠杆菌细胞的毒性较小。
同时，生物体中的 DNA 聚合酶与市售的 DNA 聚合酶不同，因此还需要在体内进行复制和转录实验。（31,47） 应考虑这些核苷酸对生理学的影响，以使用非天然核苷酸构建更健康的细胞系统。（48） 为了评估体内复制，合成了先前报道的含有 dNaM 的 D6，每侧两侧有三个随机的天然核苷酸。（16） 在 dNaM/dTPT3 存在下，使用 OneTaq 聚合酶通过 PCR 扩增模板，然后插入质粒 pBLUE-T 中。还合成了表达质粒的 PtNTT2 以转运三磷酸盐。然后，将两个质粒一起转化到大肠杆菌 BL 21 （DE3） 中。显示了工作流程（图 5A）。转化后，将大肠杆菌 BL 21 （DE3） 与 125 μM 浓度的 dNaM/dTPT3 或 dNaM/dTAT1 在 37 °C 下孵育 17 小时。最后，以大肠杆菌细胞为模板，通过 PCR 计算保留率。结果发现 dNaM-dTAT1 和 dNaM-dTPT3 在体内的保留分别为 51.4 ± 1.8 和 53.3 ± 4.4%。两个碱基对的保留率没有差异（图 5B 和图 S10）。体内复制的保留率低于之前报道的 （17），这可能是由于采用了随机序列库。众所周知，UBP 保留率与序列上下文相关，某些序列上下文显示高保留率，而另一些则较低或没有保留率。（48,49） 带有随机序列的质粒将显示综合保留。此外，E 的生长。 还通过测量 OD600 分析了大肠杆菌细胞，我们发现与 dNaM/dTAT1 一起孵育的大肠杆菌细胞显示出更强劲的生长（图 5C）。dTAT1 或 dTPT3 也与大肠杆菌细胞单独孵育。此外，大肠杆菌细胞的生长也显示出相同的趋势（图 5D）。据报道，与大肠杆菌细胞一起孵育的 UBP 可导致活性氧 （ROS） 的产生。（23） 还使用 DCFH-DA 探针监测 ROS 水平，我们发现 dTAT1 导致 ROS 水平降低（图 5E）。这些表明新的 dTAT1 对大肠杆菌细胞的毒性较小。
Figure 5 图 5
Figure 5. Intracellular UBP replication and physiology of the E. coli cells after incubation for 17 h. (A) Overview of plasmid construction and transforming. X: dNaM; Y: dTPT3; or dTAT1. Color indicates overlapped sequences. The plasmids containing UBPs or PtNTT2 were transformed into E. coli BL 21 (DE3) together. (B) Retention of the UBPs. (C) Growth of E. coli cells cultured with UBPs. (D) Growth of E. coli cells cultured with dTPT3 or dTAT1. (E) ROS content of E. coli cells cultured with dTPT3 or dTAT1. Data represents the mean ± S.D. (n = 3), Student’s t test, **p < 0.01.
图 5.孵育 17 小时后大肠杆菌细胞的细胞内 UBP 复制和生理学。（A） 质粒构建和转化概述。X： dNaM;Y： dTPT3;或 dTAT1 的 dTAT1 中。Color 表示重叠的序列。将含有 UBPs 或 PtNTT2 的质粒一起转化到大肠杆菌 BL 21 （DE3） 中。（B） 保留 UBP。（C） 用 UBP 培养的大肠杆菌细胞的生长。（D） 用 dTPT3 或 dTAT1 培养的大肠杆菌细胞的生长。（E） 用 dTPT3 或 dTAT1 培养的大肠杆菌细胞的 ROS 含量。数据表示 ± S.D. （n = 3）、学生 t 检验、**p < 0.01 的平均值。
Conclusions 结论
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In summary, we have designed and synthesized a panel of TPT3 analogues with the basic structural skeletons of TPT3 but modified thiophene rings at variant sites to improve the photostability of unnatural base pairs. A new comprehensive synthesis-screening strategy, including photosensitivity tests, kinetic experiments, and replication in vitro by PCR and in vivo by amplification, was implemented. A new pair, dNaM-dTAT1, which had almost equally high efficiency and fidelity with the dNaM-dTPT3 pair itself both in vivo and in vitro, was proven to be more photostable and thermostable and less toxic to E. coli cells. We previously have shown that the ribonucleotide homolog of TAT1 also performed excellently in transcription and translation. (10) The discovery of dTAT1TP represents our first progress for the optimization of TPT3-type bases toward more photostable properties; our data also suggest that less photosensitive unnatural base pairs will be beneficial to build a healthier cellular replication system.
总之，我们设计并合成了一组具有 TPT3 基本结构骨架的 TPT3 类似物，但在变体位点修饰了噻吩环，以提高非天然碱基对的光稳定性。实施了一种新的综合合成筛选策略，包括光敏性测试、动力学实验以及通过 PCR 在体外和通过扩增在体内复制。一种新的 dNaM-dTAT1 对在体内和体外都与 dNaM-dTPT3 对本身具有几乎相同的效率和保真度，被证明具有更高的光稳定性和热稳定性，并且对大肠杆菌细胞的毒性更小。我们之前已经表明，TAT1 的核糖核苷酸同源物在转录和翻译中也表现出色。（10） dTAT1TP 的发现代表了我们在优化 TPT3 型碱基以获得更多光稳定性方面的首次进展;我们的数据还表明，光敏感度较低的非天然碱基对将有利于构建更健康的细胞复制系统。
总之，我们设计并合成了一组具有 TPT3 基本结构骨架的 TPT3 类似物，但在变体位点修饰了噻吩环，以提高非天然碱基对的光稳定性。实施了一种新的综合合成筛选策略，包括光敏性测试、动力学实验以及通过 PCR 在体外和通过扩增在体内复制。一种新的 dNaM-dTAT1 对在体内和体外都与 dNaM-dTPT3 对本身具有几乎相同的效率和保真度，被证明具有更高的光稳定性和热稳定性，并且对大肠杆菌细胞的毒性更小。我们之前已经表明，TAT1 的核糖核苷酸同源物在转录和翻译中也表现出色。（10） dTAT1TP 的发现代表了我们在优化 TPT3 型碱基以获得更多光稳定性方面的首次进展;我们的数据还表明，光敏感度较低的非天然碱基对将有利于构建更健康的细胞复制系统。
Methods 方法
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Materials and Analytic Methods
材料和分析方法
Taq, Klenow fragment DNA polymerase I, and OneTaq NDA polymerases were purchased for New England Biolabs (Ipswich, MA). Hieff Taq DNA Polymerases were purchased from Sangon Biotech. dNTPs were purchased from Solarbio. The 134 bp single-stranded DNAs containing dNaM were purchased from GenScript, (16) and other oligonucleotides were purchased from Sangon Biotech. pBLUE-T Fast Cloning Kits and BL 21 (DE3) Electrocompetent cells were purchased from Zoman Biotechnology Co., Ltd. dNaMTP and dTPT3TP were synthesized as reported. (16,50) HRMS of all compounds was performed on a Bruker compact Ultra-high-resolution electro-spray time-of-flight mass spectrometer. MS of nucleoside triphosphates was performed on a Bruker Autoflex speed MALDLTOF/TOF spectrometer. NMR spectra were performed on a Bruker AVANCE III HD (600 MHz) or AVANCE NanoBay (400 MHz).
Taq、Klenow 片段 DNA 聚合酶 I 和 OneTaq NDA 聚合酶购自 New England Biolabs（马萨诸塞州伊普斯威奇）。Hieff Taq DNA 聚合酶购自 Sangon Biotech。dNTP 购自 Solarbio。含有 dNaM 的 134 bp 单链 DNA 购自金斯瑞 （16），其他寡核苷酸购自桑贡生物科技。pBLUE-T 快速克隆试剂盒和 BL 21 （DE3） 电感受态细胞购自 Zoman Biotechnology Co.， Ltd.。dNaMTP 和 dTPT3TP 的合成如报道所述。（16,50） 所有化合物的 HRMS 均在 Bruker 紧凑型超高分辨率电喷雾飞行时间质谱仪上进行。在 Bruker Autoflex speed MALDLTOF/TOF 光谱仪上对核苷三磷酸进行 MS。核磁共振波谱在布鲁克 AVANCE III HD （600 MHz） 或 AVANCE NanoBay （400 MHz） 上进行。
Taq、Klenow 片段 DNA 聚合酶 I 和 OneTaq NDA 聚合酶购自 New England Biolabs（马萨诸塞州伊普斯威奇）。Hieff Taq DNA 聚合酶购自 Sangon Biotech。dNTP 购自 Solarbio。含有 dNaM 的 134 bp 单链 DNA 购自金斯瑞 （16），其他寡核苷酸购自桑贡生物科技。pBLUE-T 快速克隆试剂盒和 BL 21 （DE3） 电感受态细胞购自 Zoman Biotechnology Co.， Ltd.。dNaMTP 和 dTPT3TP 的合成如报道所述。（16,50） 所有化合物的 HRMS 均在 Bruker 紧凑型超高分辨率电喷雾飞行时间质谱仪上进行。在 Bruker Autoflex speed MALDLTOF/TOF 光谱仪上对核苷三磷酸进行 MS。核磁共振波谱在布鲁克 AVANCE III HD （600 MHz） 或 AVANCE NanoBay （400 MHz） 上进行。
Photostability Assay 光稳定性测定
The absorption spectra of TPT3-type unnatural nucleosides were scanned from 200 to 900 nm using an ultraviolet spectrophotometer (TU-1900, PERSIE). Each nucleoside was dissolved in aqueous phosphate-buffered saline (pH 7.4) to obtain 0.1 M solutions. Then, equal solutions in small PE pipes (volume of sample: 200 μL) were exposed to lights with different wavelength ranges for 0.25, 0.5, 0.75, 1, and 1.25 h. LED lamps emitting near-visible (350–410 nm), red (605–740 nm), green (500–560 nm), blue (450–480 nm), or purple light (400–435 nm) were used (10 W, XINCHUANG Electronic Technology Co., Ltd). After irradiation, the samples were quantified by high-performance liquid chromatography (HPLC, UltiMate 3000, Thermo Fisher) using a C18 reverse-phase. Samples were subjected to a linear gradient of 0–30% B over 40 min at a flow rate of 1 mL/min. Buffer A: 0.1 M TEAB, pH 7.3; buffer B: acetonitrile. For TPT3 and TAT1, absorption at 365 nm was monitored; for TPT4, absorption at 346 nm was monitored; and for d4TFP, absorption at 327 nm was monitored. The data are averages and standard deviations of three independent determinations.
使用紫外分光光度计 （TU-1900， PERSIE） 从 200 到 900 nm 扫描 TPT3 型非天然核苷的吸收光谱。将每种核苷溶于磷酸盐缓冲盐水 （pH 7.4） 中，得到 0.1 M 溶液。然后，将小 PE 管（样品体积：200 μL）中的等溶液暴露在不同波长范围的光下 0.25、0.5、0.75、1 和 1.25 h。使用发射近可见光 （350–410 nm）、红光 （605–740 nm）、绿光 （500–560 nm）、蓝光 （450–480 nm） 或紫光 （400–435 nm） 的 LED 灯 （10 W， 鑫创电子科技有限公司）。照射后，使用 C18 反相通过高效液相色谱（HPLC、UltiMate 3000、Thermo Fisher）对样品进行定量。样品以 1 mL/min 的流速在 40 分钟内以 0–30% B 的线性梯度进行实验。缓冲液 A：0.1 M TEAB，pH 7.3;缓冲液 B：乙腈。对于 TPT3 和 TAT1，监测 365 nm 处的吸收;对于 TPT4，监测 346 nm 处的吸收;对于 d4TFP，监测 327 nm 处的吸收。数据是三个独立测定的平均值和标准差。
使用紫外分光光度计 （TU-1900， PERSIE） 从 200 到 900 nm 扫描 TPT3 型非天然核苷的吸收光谱。将每种核苷溶于磷酸盐缓冲盐水 （pH 7.4） 中，得到 0.1 M 溶液。然后，将小 PE 管（样品体积：200 μL）中的等溶液暴露在不同波长范围的光下 0.25、0.5、0.75、1 和 1.25 h。使用发射近可见光 （350–410 nm）、红光 （605–740 nm）、绿光 （500–560 nm）、蓝光 （450–480 nm） 或紫光 （400–435 nm） 的 LED 灯 （10 W， 鑫创电子科技有限公司）。照射后，使用 C18 反相通过高效液相色谱（HPLC、UltiMate 3000、Thermo Fisher）对样品进行定量。样品以 1 mL/min 的流速在 40 分钟内以 0–30% B 的线性梯度进行实验。缓冲液 A：0.1 M TEAB，pH 7.3;缓冲液 B：乙腈。对于 TPT3 和 TAT1，监测 365 nm 处的吸收;对于 TPT4，监测 346 nm 处的吸收;对于 d4TFP，监测 327 nm 处的吸收。数据是三个独立测定的平均值和标准差。
Gel-Based Incorporation/Extension Assay
基于凝胶的掺入/延伸检测
Templates and HEX-labeled primers (Sangon Biotech) were annealed by heating to 95 °C and then slowly cooled to room temperature (Sequence Context II in Seo, et al. (50)). Reactions were initiated by adding 2 × dYTP or 2 × dYTP and dCTP solution (5 μL) to a 5 μL solution containing 0.3 μM primer/template and 0.44 μM Klenow fragment DNA polymerase I (Kf, NEB). All solutions were in Kf buffer (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9). For the incorporation assay, 12.5 μM dYTP was added. For the extension assay, 12.5 μM dYTP and dCTP were added. Reactions were incubated at 25 °C for 10 s and quenched with 20 μL loading dye (90% formamide, 30 mM EDTA, and sufficient amounts of bromophenol blue and xylene cyanol). Finally, the full length was obtained by adding 6.25 μM dYTP and 12.5 μM four natural bases. Reactions were incubated at 25 °C for 15 min and then quenched with 20 μL of loading dye. The reaction mix was analyzed by 8 M urea 15% denaturing polyacrylamide gel electrophoresis, and fluorescence intensities were quantified by fluorescence imaging (Amersham Imager 680, AI600 software). The data are averages and standard deviations of three independent determinations.
模板和 HEX 标记的引物 （Sangon Biotech） 通过加热至 95 °C 进行退火，然后缓慢冷却至室温（Seo 等人的序列上下文 II。（50））. 将 2 × dYTP 或 2 × dYTP 和 dCTP 溶液 （5 μL） 加入到含有 0.3 μM 引物/模板和 0.44 μM Klenow 片段 DNA 聚合酶 I （Kf， NEB） 的 5 μL 溶液中，引发反应。所有溶液均溶于 Kf 缓冲液（50 mM NaCl、10 mM Tris-HCl、10 mM MgCl2、1 mM DTT，pH 7.9）中。对于掺入测定，添加了 12.5 μM dYTP。对于延伸测定，加入 12.5 μM dYTP 和 dCTP。将反应物在 25 °C 下孵育 10 秒，并用 20 μL 上样染料（90% 甲酰胺、30 mM EDTA 和足量的溴酚蓝和二甲苯蓝）淬灭。最后，通过添加 6.25 μM dYTP 和 12.5 μM 四种天然碱基获得全长。将反应物在 25 °C 下孵育 15 分钟，然后用 20 μL 上样染料淬灭。通过 8 M 尿素 15% 变性聚丙烯酰胺凝胶电泳分析反应混合物，并通过荧光成像 （Amersham Imager 680， AI600 软件） 定量荧光强度。数据是三个独立测定的平均值和标准差。
模板和 HEX 标记的引物 （Sangon Biotech） 通过加热至 95 °C 进行退火，然后缓慢冷却至室温（Seo 等人的序列上下文 II。（50））. 将 2 × dYTP 或 2 × dYTP 和 dCTP 溶液 （5 μL） 加入到含有 0.3 μM 引物/模板和 0.44 μM Klenow 片段 DNA 聚合酶 I （Kf， NEB） 的 5 μL 溶液中，引发反应。所有溶液均溶于 Kf 缓冲液（50 mM NaCl、10 mM Tris-HCl、10 mM MgCl2、1 mM DTT，pH 7.9）中。对于掺入测定，添加了 12.5 μM dYTP。对于延伸测定，加入 12.5 μM dYTP 和 dCTP。将反应物在 25 °C 下孵育 10 秒，并用 20 μL 上样染料（90% 甲酰胺、30 mM EDTA 和足量的溴酚蓝和二甲苯蓝）淬灭。最后，通过添加 6.25 μM dYTP 和 12.5 μM 四种天然碱基获得全长。将反应物在 25 °C 下孵育 15 分钟，然后用 20 μL 上样染料淬灭。通过 8 M 尿素 15% 变性聚丙烯酰胺凝胶电泳分析反应混合物，并通过荧光成像 （Amersham Imager 680， AI600 软件） 定量荧光强度。数据是三个独立测定的平均值和标准差。
PCR assay PCR 检测
All PCRs were performed with different DNA polymerases in a volume of 25 μL under the following conditions: 1 × reaction buffer with DNA polymerases, 0.2 mM dNTP, 0.1 mM dNaMTP and dTPT3TP or its analogues, 0.04 ng of the 134-mer templates or 2 μL bacterial fluid, and 1 μM primers under the following thermal cycling conditions: initial denaturation (96 °C, 1 min) followed by 16, 20, 36 or 3 × 17 cycles of denaturation (96 °C, 10 s), annealing (60 °C, 15 s), and extension (68 °C, 1 min), and then another extension (68 °C, 5 min). The PCR products were detected by 2% agarose gel and sequenced by Sangon Biotech. The percent retention of an unnatural base pair (F) was calculated using the raw sequencing data, and the fidelities were determined as the average % retention (F) of the unnatural base pair per doubling as reported. (50,51) Due to the significant read-through in the F direction, only the other direction (R) was used to gauge PCR fidelity without correcting or removing the read-through, which would reduce the retention value but did not affect the comparison between dTPT3-dNaM and its analogues-dNaM. The data are averages and standard deviations of three independent determinations.
在以下条件下，使用 25 μL 体积的不同 DNA 聚合酶进行所有 PCR：在以下热循环条件下，使用 1 × 反应缓冲液，加入 DNA 聚合酶、0.2 mM dNTP、0.1 mM dNaMTP 和 dTPT3TP 或其类似物、0.04 ng 134 mer 模板或 2 μL 细菌液和 1 μM 引物： 初始变性（96°C，1分钟），然后是16、20、36或3×17个变性循环（96°C，10秒），退火（60°C，15秒）和延伸（68°C，1分钟），然后是另一个延伸（68°C，5分钟）。用 2% 琼脂糖凝胶检测 PCR 产物，并由 Sangon Biotech 测序。使用原始测序数据计算非天然碱基对的保留百分比 （F），并将保真度确定为报告的每次倍增非天然碱基对的平均保留百分比 （F）。（50,51） 由于 F 方向的显著通读，仅使用另一个方向 （R） 来测量 PCR 保真度，而没有校正或删除通读，这会降低保留值，但不会影响 dTPT3-dNaM 与其类似物-dNaM 之间的比较。数据是三个独立测定的平均值和标准差。
在以下条件下，使用 25 μL 体积的不同 DNA 聚合酶进行所有 PCR：在以下热循环条件下，使用 1 × 反应缓冲液，加入 DNA 聚合酶、0.2 mM dNTP、0.1 mM dNaMTP 和 dTPT3TP 或其类似物、0.04 ng 134 mer 模板或 2 μL 细菌液和 1 μM 引物： 初始变性（96°C，1分钟），然后是16、20、36或3×17个变性循环（96°C，10秒），退火（60°C，15秒）和延伸（68°C，1分钟），然后是另一个延伸（68°C，5分钟）。用 2% 琼脂糖凝胶检测 PCR 产物，并由 Sangon Biotech 测序。使用原始测序数据计算非天然碱基对的保留百分比 （F），并将保真度确定为报告的每次倍增非天然碱基对的平均保留百分比 （F）。（50,51） 由于 F 方向的显著通读，仅使用另一个方向 （R） 来测量 PCR 保真度，而没有校正或删除通读，这会降低保留值，但不会影响 dTPT3-dNaM 与其类似物-dNaM 之间的比较。数据是三个独立测定的平均值和标准差。
Computational Details 计算详细信息
The nucleotides were designed as free bases terminated by a methyl group in place of the sugar phosphate backbone. Their respective pairs were optimized within a DFT approach implemented in the Gaussian 09 package. The quantum chemical geometry and interaction energies were calculated at the B3LYP/6-31 + G(d) level of theory in the gas phase. (52,53) The electrostatic potential (ESP) maps described the electron density distribution; the red area represents the electron-rich area; and the green area represents the electron-deficient area.
核苷酸被设计为以甲基为末端的游离碱基，以代替磷酸糖骨架。它们各自的对在 Gaussian 09 包中实现的 DFT 方法中进行了优化。在气相中，量子化学几何和相互作用能在 B3LYP/6-31 + G（d） 理论水平上计算。（52,53） 静电势 （ESP） 图描述了电子密度分布;红色区域代表电子富集区域;绿色区域代表缺电子区域。
核苷酸被设计为以甲基为末端的游离碱基，以代替磷酸糖骨架。它们各自的对在 Gaussian 09 包中实现的 DFT 方法中进行了优化。在气相中，量子化学几何和相互作用能在 B3LYP/6-31 + G（d） 理论水平上计算。（52,53） 静电势 （ESP） 图描述了电子密度分布;红色区域代表电子富集区域;绿色区域代表缺电子区域。
Growth and In Vivo Replication Assay
生长和体内复制检测
The plasmid-expressed PtNTT2 was synthesized according to the previous report (48) by GenScript. Also, the 134-mer template containing dNaM flanked on each side by three randomized natural nucleotides (D6 (16)) was amplified by PCR with OneTaq. A linear fragment was amplified from pBLUE-T. Then, the products were purified by the DNA gel Recovery Kit from TsingKe. The linear fragment and 134-mer template were combined and assembled by circular overlap extension PCR based on literature methods (2) under the following thermocycling conditions: [96 °C, 1 min | 20× (96 °C, 30 s| 60 °C, 15 s | 68 °C, 4 min) | 68 °C, 5 min]. The PCR product was analyzed by restriction digestion and used directly for E. coli transformation.
质粒表达的 PtNTT2 由金斯瑞根据之前的报告 （48） 合成。此外，使用 OneTaq 通过 PCR 扩增含有 dNaM 且两侧各有三个随机天然核苷酸 （D6 （16）） 的 134 聚体模板。从 pBLUE-T 扩增线性片段。然后，使用 Tsingke 的 DNA 凝胶回收试剂盒纯化产物。在以下热循环条件下，将线性片段和 134 聚体模板合并并通过基于文献方法 （2） 的环重叠延伸 PCR 进行组装：[96 °C，1 分钟 | 20×（96 °C，30 s| 60 °C，15 秒 | 68 °C，4 分钟）| 68 °C，5 分钟]。通过限制性酶切分析 PCR 产物，并直接用于大肠杆菌转化。
质粒表达的 PtNTT2 由金斯瑞根据之前的报告 （48） 合成。此外，使用 OneTaq 通过 PCR 扩增含有 dNaM 且两侧各有三个随机天然核苷酸 （D6 （16）） 的 134 聚体模板。从 pBLUE-T 扩增线性片段。然后，使用 Tsingke 的 DNA 凝胶回收试剂盒纯化产物。在以下热循环条件下，将线性片段和 134 聚体模板合并并通过基于文献方法 （2） 的环重叠延伸 PCR 进行组装：[96 °C，1 分钟 | 20×（96 °C，30 s| 60 °C，15 秒 | 68 °C，4 分钟）| 68 °C，5 分钟]。通过限制性酶切分析 PCR 产物，并直接用于大肠杆菌转化。
The two plasmids were transformed to BL 21 (DE3) by electroporation (Gene Pulser II; Bio-Rad) according to the previous report (2) (voltage 25 kV, capacitor 2.5 μF, resistor 200 Ω, time constant 5 ms). The cells were then centrifuged at 2400 rpm for 10 min at 4 °C to remove the culture. Also, the cells were resuspended with 2 × YT media (100 μL 2 × YT media (casein peptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L), 25 μM chloramphenicol, 100 μg/mL ampicillin, 50 mM KPi) with 125 μM dTPT3 and dNaM and incubated at 37 °C for 40 min. Then, the cells were centrifuged at 2400 rpm for 10 min at 4 °C and resuspended with 2 × YT media (100 μL, 25 μM chloramphenicol, 100 μg/mL ampicillin, 50 mM KPi). Finally, 5 μL supernatant was added to the 2 × YT culture (100 μL, 25 μM chloramphenicol, 100 μg/mL ampicillin, 50 mM KPi) with 125 μM dNaM/dTPT3 or dNaM/dTAT1, incubating at 37 °C for 17 h. The growth of bacteria (OD600) was monitored by NanoDrop OneC and then used for the PCR assay.
通过电穿孔将两个质粒转化为 BL 21 （DE3） （Gene Pulser II;Bio-Rad）根据之前的报告 （2）（电压 25 kV，电容器 2.5 μF，电阻器 200 Ω，时间常数 5 ms）。然后将细胞在 4 °C 下以 2400 rpm 离心 10 分钟以去除培养物。此外，用 2 × YT 培养基（100 μL 2 × YT 培养基（酪蛋白蛋白胨 16 g/L、酵母提取物 10 g/L、NaCl 5 g/L）、25 μM 氯霉素、100 μg/mL 氨苄青霉素、50 mM KPi）和 125 μM dTPT3 和 dNaM 重悬细胞，并在 37 °C 下孵育 40 分钟。然后，将细胞在 4 °C 下以 2400 rpm 离心 10 分钟，并用 2 × YT 培养基（100 μL、25 μM 氯霉素、100 μg/mL 氨苄青霉素、50 mM KPi）重悬。最后，将 5 μL 上清液加入 2 × YT 培养物（100 μL、25 μM 氯霉素、100 μg/mL 氨苄青霉素、50 mM KPi）和 125 μM dNaM/dTPT3 或 dNaM/dTAT1 中，在 37 °C 下孵育 17 小时。通过 NanoDrop OneC 监测细菌 （OD600） 的生长，然后用于 PCR 测定。
通过电穿孔将两个质粒转化为 BL 21 （DE3） （Gene Pulser II;Bio-Rad）根据之前的报告 （2）（电压 25 kV，电容器 2.5 μF，电阻器 200 Ω，时间常数 5 ms）。然后将细胞在 4 °C 下以 2400 rpm 离心 10 分钟以去除培养物。此外，用 2 × YT 培养基（100 μL 2 × YT 培养基（酪蛋白蛋白胨 16 g/L、酵母提取物 10 g/L、NaCl 5 g/L）、25 μM 氯霉素、100 μg/mL 氨苄青霉素、50 mM KPi）和 125 μM dTPT3 和 dNaM 重悬细胞，并在 37 °C 下孵育 40 分钟。然后，将细胞在 4 °C 下以 2400 rpm 离心 10 分钟，并用 2 × YT 培养基（100 μL、25 μM 氯霉素、100 μg/mL 氨苄青霉素、50 mM KPi）重悬。最后，将 5 μL 上清液加入 2 × YT 培养物（100 μL、25 μM 氯霉素、100 μg/mL 氨苄青霉素、50 mM KPi）和 125 μM dNaM/dTPT3 或 dNaM/dTAT1 中，在 37 °C 下孵育 17 小时。通过 NanoDrop OneC 监测细菌 （OD600） 的生长，然后用于 PCR 测定。
The dTPT3 and dTAT1 were also incubated with BL 21 (DE3) alone for 17 h. The growth (OD600) was detected. Then, the level of reactive oxygen species (ROS) in the whole cultured system was monitored by the kit from Nanjing Jiancheng according to the manufacturer’s recommendation. The cells were incubated with DCFH-DA (2,7-dichlorodi-hydrofluorescein diacetate) at 37 °C for 30 min, and the level of ROS was detected by a multifunctional microplate reader (EnVision, PerkinElmer). The data are averages and standard deviations of three independent determinations.
dTPT3 和 dTAT1 也与 BL 21 （DE3） 单独孵育 17 小时。检测到生长 （OD600）。然后，根据制造商的建议，用南京建成的试剂盒监测整个培养系统中的活性氧 （ROS） 水平。将细胞与 DCFH-DA （2,7-二氯二氢荧光素二乙酸酯） 在 37 °C 下孵育 30 分钟，并通过多功能酶标仪 （EnVision， PerkinElmer） 检测 ROS 水平。数据是三个独立测定的平均值和标准差。
dTPT3 和 dTAT1 也与 BL 21 （DE3） 单独孵育 17 小时。检测到生长 （OD600）。然后，根据制造商的建议，用南京建成的试剂盒监测整个培养系统中的活性氧 （ROS） 水平。将细胞与 DCFH-DA （2,7-二氯二氢荧光素二乙酸酯） 在 37 °C 下孵育 30 分钟，并通过多功能酶标仪 （EnVision， PerkinElmer） 检测 ROS 水平。数据是三个独立测定的平均值和标准差。
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Author Information 作者信息
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- Lingjun Li - Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, China;
https://orcid.org/0000-0002-4722-3637;
李玲军 - 河南师范大学 河南省有机功能分子与药物创新重点实验室， 河南省精细化工绿色制造协同创新中心， 河南省化学化工学院， 绿色化学介质与反应教育部重点实验室， 河南 453007 新乡; https://orcid.org/0000-0002-4722-3637;
- Nana Ma - Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, China;https://orcid.org/0000-0003-3225-9554
马河南师范大学 河南省有机功能分子与药物创新重点实验室， 河南省精细化工绿色制造协同创新中心， 河南师范大学化学化工学院， 绿色化学介质与反应教育部重点实验室， 河南师范大学， 河南省 新乡， 河南 453007; https://orcid.org/0000-0003-3225-9554
H.W., L.W., and N.M. contributed equally to this work.
H.W. and L.L. conceived the projects and designed the experiments. L.W., N.M., W.Z., B.H., and A.Z. performed the experiments. All authors contributed to the data analysis and manuscript preparation.
Acknowledgments
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This work was funded by the National Natural Science Foundation of China (22077027, 21778015 to L.L.), Central Plains Science and Technology Innovation Leader Project (214200510008 to L.L.), Doctoral Initiation Fund (31901014, qd18008 to H.W., 5101039470644 to B.H.), and 21IRTSTHN001.
References
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This article references 53 other publications.
- 1Piccirilli, J. A.; Benner, S. A.; Krauch, T.; Moroney, S. E. Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature 1990, 343, 33– 37, DOI: 10.1038/343033a0Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXitFamu7k%253D&md5=1abf50139303a7ffa31262cdeb736fe5Enzymic incorporation of a new base pair into DNA and RNA extends the genetic alphabetPiccirilli, Joseph A.; Krauch, Tilman; Moroney, Simon E.; Benner, Steven A.Nature (London, United Kingdom) (1990), 343 (6253), 33-7CODEN: NATUAS; ISSN:0028-0836.A new Watson-Crick base pair [κ-xanthine or 7-Me oxyformycin B, where κ = 3-β-D-ribofuranosyl-(2,6-diaminopyrimidine) with a H bonding pattern different from that in the A·T and G·C base pairs, is incorporated into duplex DNA and RNA by DNA and RNA polymerases and expands the genetic alphabet from 4 to 6 letters. This expansion could lead to RNAs with greater diversity in functional groups and greater catalytic potential.
- 2Malyshev, D. A.; Dhami, K.; Lavergne, T.; Chen, T.; Dai, N.; Foster, J. M.; Corrêa, I. R.; Romesberg, F. E. A semi-synthetic organism with an expanded genetic alphabet. Nature 2014, 509, 385– 388, DOI: 10.1038/nature13314Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXotVyqtb8%253D&md5=97b4b184cda52cc809b1705e5e88ad8eA semi-synthetic organism with an expanded genetic alphabetMalyshev, Denis A.; Dhami, Kirandeep; Lavergne, Thomas; Chen, Tingjian; Dai, Nan; Foster, Jeremy M.; Correa, Ivan R.; Romesberg, Floyd E.Nature (London, United Kingdom) (2014), 509 (7500), 385-388CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA contg. the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid contg. d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
- 3Kimoto, M.; Hirao, I. Genetic alphabet expansion technology by creating unnatural base pairs. Chem. Soc. Rev. 2020, 49, 7602– 7626, DOI: 10.1039/D0CS00457JGoogle Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFyit7bL&md5=8bb0ea148d02d47f184b412bd0e616feGenetic alphabet expansion technology by creating unnatural base pairsKimoto, Michiko; Hirao, IchiroChemical Society Reviews (2020), 49 (21), 7602-7626CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Recent advancements in the creation of artificial extra base pairs (unnatural base pairs, UBPs) are opening the door to a new research area, xenobiol., and genetic alphabet expansion technologies. UBPs that function as third base pairs in replication, transcription, and/or translation enable the site-specific incorporation of novel components into DNA, RNA, and proteins. Here, we describe the UBPs developed by three research teams and their application in PCR-based diagnostics, high-affinity DNA aptamer generation, site-specific labeling of RNAs, semi-synthetic organism creation, and unnatural-amino-acid-contg. protein synthesis.
- 4Ledbetter, M. P.; Malyshev, D. A.; Romesberg, F. E. Site-Specific Labeling of DNA via PCR with an Expanded Genetic Alphabet. Methods Mol. Biol. 2019, 1973, 193– 212, DOI: 10.1007/978-1-4939-9216-4_13Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVWrurk%253D&md5=a793c10929b681f4e69b91210933647aSite-specific labeling of DNA via PCR with an expanded genetic alphabetLedbetter, Michael P.; Malyshev, Denis A.; Romesberg, Floyd E.Methods in Molecular Biology (New York, NY, United States) (2019), 1973 (Non-Natural Nucleic Acids), 193-212CODEN: MMBIED; ISSN:1940-6029. (Springer)A review. The polymerase chain reaction (PCR) is a universal and essential tool in mol. biol. and biotechnol., but it is generally limited to the amplification of DNA with the four-letter genetic alphabet. Here, we describe PCR amplification with a six-letter alphabet that includes the two natural dA-dT and dG-dC base pairs and an unnatural base pair (UBP) formed between the synthetic nucleotides dNaM and d5SICS or dTPT3 or analogs of these synthetic nucleotides modified with linkers that allow for the site-specific labeling of the amplified DNA with different functional groups. Under std. conditions, the six-letter DNA may be amplified with high efficiency and with greater than 99.9% fidelity. This allows for the efficient prodn. of DNA site-specifically modified with different functionalities of interest for use in a wide range of applications.
- 5Kimoto, M.; Mitsui, T.; Harada, Y.; Sato, A.; Yokoyama, S.; Hirao, I. Fluorescent probing for RNA molecules by an unnatural base-pair system. Nucleic Acids Res. 2007, 35, 5360– 5369, DOI: 10.1093/nar/gkm508Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1ygtb7N&md5=9229005556346b52fa5f42fba40a7200Fluorescent probing for RNA molecules by an unnatural base-pair systemKimoto, Michiko; Mitsui, Tsuneo; Harada, Yoko; Sato, Akira; Yokoyama, Shigeyuki; Hirao, IchiroNucleic Acids Research (2007), 35 (16), 5360-5369CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl) purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5'-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates. The fluorescent intensity of s in RNA mols. changes according to the structural environment. The site-specific s labeling of RNA hairpins and tRNA mols. provided characteristic fluorescent profiles, depending on the labeling sites, temp. and Mg2+ concn. The Pa-contg. DNA templates can be amplified by PCR using 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), another pairing partner of Pa. This site-specific fluorescent probing by the unnatural pair system including the s-Pa and Ds-Pa pairs provides a powerful tool for studying the dynamics of the local structural features of 3D RNA mols. and their intra- and intermol. interactions.
- 6Kimoto, M.; Matsunaga, K. I.; Hirao, I. Evolving Aptamers with Unnatural Base Pairs. Curr. Protoc. Chem. Biol. 2017, 9, 315– 339, DOI: 10.1002/cpch.31Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjtVOmsrw%253D&md5=5d47ec6b5ec780052ef39872705a9f42Evolving Aptamers with Unnatural Base PairsKimoto, Michiko; Matsunaga, Ken-ichiro; Hirao, IchiroCurrent Protocols in Chemical Biology (2017), 9 (4), 315-339CODEN: CPCBD9; ISSN:2160-4762. (John Wiley & Sons, Inc.)A novel technol., genetic alphabet expansion, has rapidly advanced through the successful creation of unnatural base pairs that function as a third base pair in replication. Recently, genetic alphabet expansion has been applied to some practical areas. Among them, the application to DNA aptamer generation is a good example of the broad utility of this technol. A hydrophobic unnatural base pair, Ds-Px, which exhibits high fidelity in replication as a third base pair, has been applied to an evolutionary engineering method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) to generate DNA aptamers that bind to targets. A few Ds bases in DNA aptamers significantly increase the binding affinity to targets, enabling the use of DNA aptamers as an alternative to antibodies. This protocol describes the ExSELEX (genetic alphabet Expansion for SELEX) method to generate Ds-contg. DNA aptamers. © 2017 by John Wiley & Sons, Inc.
- 7Hoshika, S.; Leal, N. A.; Kim, M.-J.; Kim, M.-S.; Karalkar, N. B.; Kim, H.-J.; Bates, A. M.; Watkins, N. E., Jr.; SantaLucia, H. A.; Meyer, A. J.; DasGupta, S.; Piccirilli, J. A.; Ellington, A. D.; SantaLucia, J., Jr.; Georgiadis, M. M.; Benner, S. A. Hachimoji DNA and RNA: a genetic system with eight building blocks. Science 2019, 363, 884– 887, DOI: 10.1126/science.aat0971Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjt1elu7c%253D&md5=1ae87a691cfdfbe36a819d4782c305b9Hachimoji DNA and RNA: A genetic system with eight building blocksHoshika, Shuichi; Leal, Nicole A.; Kim, Myong-Jung; Kim, Myong-Sang; Karalkar, Nilesh B.; Kim, Hyo-Joong; Bates, Alison M.; Watkins, Norman E., Jr.; SantaLucia, Holly A.; Meyer, Adam J.; DasGupta, Saurja; Piccirilli, Joseph A.; Ellington, Andrew D.; SantaLucia, John, Jr.; Georgiadis, Millie M.; Benner, Steven A.Science (Washington, DC, United States) (2019), 363 (6429), 884-887CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodn. stability, and stereoregular building blocks that fit a Schroedinger aperiodic crystal. Measured thermodn. parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information d. of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of mol. structures that might support life, including life throughout the cosmos.
- 8Kimoto, M.; Yamashige, R.; Matsunaga, K.; Yokoyama, S.; Hirao, I. Generation of high-affinity DNA aptamers using an expanded genetic alphabet. Nat. Biotechnol. 2013, 31, 453– 457, DOI: 10.1038/nbt.2556Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltlOhur4%253D&md5=068493fd06e1537c468ed340055ce3ecGeneration of high-affinity DNA aptamers using an expanded genetic alphabetKimoto, Michiko; Yamashige, Rie; Matsunaga, Ken-ichiro; Yokoyama, Shigeyuki; Hirao, IchiroNature Biotechnology (2013), 31 (5), 453-457CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)DNA aptamers produced with natural or modified natural nucleotides often lack the desired binding affinity and specificity to target proteins. Here we describe a method for selecting DNA aptamers contg. the four natural nucleotides and an unnatural nucleotide with the hydrophobic base 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). We incorporated up to three Ds nucleotides in a random sequence library, which is expected to increase the chem. and structural diversity of the DNA mols. Selection expts. against two human target proteins, vascular endothelial cell growth factor-165 (VEGF-165) and interferon-γ (IFN-γ), yielded DNA aptamers that bind with KD values of 0.65 pM and 0.038 nM, resp., affinities that are >100-fold improved over those of aptamers contg. only natural bases. These results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids.
- 9Manandhar, M.; Chun, E.; Romesberg, F. E. Genetic code expansion: inception, development, commercialization. J. Am. Chem. Soc. 2021, 143, 4859– 4878, DOI: 10.1021/jacs.0c11938Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmvFarsb0%253D&md5=013699b4fc4a239c1424b6d7786e6eb5Genetic Code Expansion: Inception, Development, CommercializationManandhar, Miglena; Chun, Eugene; Romesberg, Floyd E.Journal of the American Chemical Society (2021), 143 (13), 4859-4878CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A review. Virtually all natural proteins are built from only 20 amino acids, and while this makes possible all the functions they perform, the ability to encode other amino acids selected for specific purposes promises to enable the discovery and prodn. of proteins with novel functions, including therapeutic proteins with more optimal drug-like properties. The field of genetic code expansion (GCE) has for years enabled the prodn. of such proteins for academic purposes and is now transitioning to commercialization for the prodn. of more optimal protein therapeutics. Focusing on E. coli, we review the history and current state of the field. We also provide a review of the first generation commercialization efforts, the lessons learned, and how those lessons are guiding new efforts. With continued academic and industrial progress, GCE methodologies promise to make possible the routine optimization of proteins for therapeutic use in a way that has only previously been possible with small-mol. therapeutics.
- 10Feldman, A. W.; Dien, V. T.; Karadeema, R. J.; Fischer, E. C.; You, Y.; Anderson, B. A.; Krishnamurthy, R.; Chen, J. S.; Li, L.; Romesberg, F. E. Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism. J. Am. Chem. Soc. 2019, 141, 10644– 10653, DOI: 10.1021/jacs.9b02075Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1eru7%252FL&md5=546bdf9cea64eed550829034588078caOptimization of Replication, Transcription, and Translation in a Semi-Synthetic OrganismFeldman, Aaron W.; Dien, Vivian T.; Karadeema, Rebekah J.; Fischer, Emil C.; You, Yanbo; Anderson, Brooke A.; Krishnamurthy, Ramanarayanan; Chen, Jason S.; Li, Lingjun; Romesberg, Floyd E.Journal of the American Chemical Society (2019), 141 (27), 10644-10653CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins contg. noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogs. Importantly, this includes the first in vivo structure-activity relationship (SAR) anal. of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were obsd., and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homol. to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins contg. multiple, proximal ncAAs.
- 11Zhou, A. X.-Z.; Sheng, K.; Feldman, A. W.; Romesberg, F. E. Progress toward Eukaryotic Semisynthetic Organisms: Translation of Unnatural Codons. J. Am. Chem. Soc. 2019, 141, 20166– 20170, DOI: 10.1021/jacs.9b09080Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlymtrzI&md5=f27e1a7d3a75e53648b2fdc724f46a47Progress toward Eukaryotic Semisynthetic Organisms: Translation of Unnatural CodonsZhou, Anne Xiao-Zhou; Sheng, Kai; Feldman, Aaron W.; Romesberg, Floyd E.Journal of the American Chemical Society (2019), 141 (51), 20166-20170CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have created a bacterial semisynthetic organism (SSO) that retains an unnatural base pair (UBP) in its DNA, transcribes it into mRNA and tRNA with cognate unnatural codons and anticodons, and after the tRNA is charged with a noncanonical amino acid, synthesizes proteins contg. the noncanonical amino acid. Here, we report the first progress toward the creation of eukaryotic SSOs. After demonstrating proof-of-concept with human HEK293 cells, we show that a variety of different unnatural codon-anticodon pairs can efficiently mediate the synthesis of unnatural proteins in CHO cells. Interestingly, we find that there are both similarities and significant differences between how the prokaryotic and eukaryotic ribosomes recognize the UBP, with the eukaryotic ribosome appearing more tolerant. The results represent the first progress toward eukaryotic SSOs and in fact, suggest that such SSOs might be able to retain more unnatural information than their bacterial counterparts.
- 12Fischer, E. C.; Hashimoto, K.; Zhang, Y.; Feldman, A. W.; Dien, V. T.; Karadeema, R. J.; Adhikary, R.; Ledbetter, M. P.; Krishnamurthy, R.; Romesberg, F. E. New codons for efficient production of unnatural proteins in a semisynthetic organism. Nat. Chem. Biol. 2020, 16, 570– 576, DOI: 10.1038/s41589-020-0507-zGoogle Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsFShsL4%253D&md5=567a5f0d421422c14ff754fa23e30f84New codons for efficient production of unnatural proteins in a semisynthetic organismFischer, Emil C.; Hashimoto, Koji; Zhang, Yorke; Feldman, Aaron W.; Dien, Vivian T.; Karadeema, Rebekah J.; Adhikary, Ramkrishna; Ledbetter, Michael P.; Krishnamurthy, Ramanarayanan; Romesberg, Floyd E.Nature Chemical Biology (2020), 16 (5), 570-576CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Abstr.: Natural organisms use a four-letter genetic alphabet that makes available 64 triplet codons, of which 61 are sense codons used to encode proteins with the 20 canonical amino acids. We have shown that the unnatural nucleotides dNaM and dTPT3 can pair to form an unnatural base pair (UBP) and allow for the creation of semisynthetic organisms (SSOs) with addnl. sense codons. Here, we report a systematic anal. of the unnatural codons. We identify nine unnatural codons that can produce unnatural protein with nearly complete incorporation of an encoded noncanonical amino acid (ncAA). We also show that at least three of the codons are orthogonal and can be simultaneously decoded in the SSO, affording the first 67-codon organism. The ability to incorporate multiple, different ncAAs site specifically into a protein should now allow the development of proteins with novel activities, and possibly even SSOs with new forms and functions. [graphic not available: see full text]. The GenBank accesion nos. were added from NCBI database MN882182-MN882190.
- 13Hamashima, K.; Kimoto, M.; Hirao, I. Creation of unnatural base pairs for genetic alphabet expansion toward synthetic xenobiology. Curr. Opin. Chem. Biol. 2018, 46, 108– 114, DOI: 10.1016/j.cbpa.2018.07.017Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVSmtLfJ&md5=9d539f07e70cccfa4372d13fc3c026f4Creation of unnatural base pairs for genetic alphabet expansion toward synthetic xenobiologyHamashima, Kiyofumi; Kimoto, Michiko; Hirao, IchiroCurrent Opinion in Chemical Biology (2018), 46 (), 108-114CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Artificial extra base pairs (unnatural base pairs, UBPs) expand the genetic alphabet of DNA, thus broadening entire biol. systems in the central dogma. UBPs function as third base pairs in replication, transcription, and/or translation, and have created a new research area, synthetic xenobiol., providing genetic engineering tools to generate novel DNAs, RNAs, and proteins with increased functionalities. Several UBPs have been developed and applied to PCR technol., DNA aptamer generation, and semi-synthetic organism creation. Among them, we developed a series of UBPs and demonstrated unique quant. PCR and high-affinity DNA aptamer generation methods.
- 14Saito-Tarashima, N.; Minakawa, N. Unnatural Base Pairs for Synthetic Biology. Chem. Pharm. Bull. 2018, 66, 132– 138, DOI: 10.1248/cpb.c17-00685Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVKrtrbF&md5=0bb731d9dec57019c159f4b02b35bdacUnnatural base pairs for synthetic biologySaito-Tarashima, Noriko; Minakawa, NoriakiChemical & Pharmaceutical Bulletin (2018), 66 (2), 132-138CODEN: CPBTAL; ISSN:0009-2363. (Pharmaceutical Society of Japan)A review. In this review, we have summarized the research effort into the development of unnatural base pairs beyond std. Watson-Crick (WC) base pairs for synthetic biol. Prior to introducing our research results, we present investigations by four outstanding groups in the field. Their research results demonstrate the importance of shape complementarity and stacking ability as well as hydrogen-bonding (H-bonding) pat- terns for unnatural base pairs. On the basis of this research background, we developed unnatural base pairs consisting of imidazo[5'4':4.5]pyrido[2,3-d]pyrimidines and 1,8-naphthyridines, i.e., I in : Na pairs. Since Im bases are recognized as ring-expanded purines and Na bases are recognized as ring-expanded pyrimidines, I m :Na pairs are expected to satisfy the criteria of shape complementarity and enhanced stacking ability. In addn., these pairs have four non-canonical H-bonds. Because of these preferable properties, ImNN:NaOO, one of the Im:Na pairs, is recognized as a complementary base pair in not only single nucleotide insertion, but also the PCR.
- 15Marx, A.; Betz, K. The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases. Chem. – Eur. J. 2020, 26, 3446– 3463, DOI: 10.1002/chem.201903525Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1Gqsrs%253D&md5=7ef06701ea4cb58153a780f96ef717c4The Structural Basis for Processing of Unnatural Base Pairs by DNA PolymerasesMarx, Andreas; Betz, KarinChemistry - A European Journal (2020), 26 (16), 3446-3463CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Unnatural base pairs (UBPs) greatly increase the diversity of DNA and RNA, furthering their broad range of mol. biol. and biotechnol. approaches. Different candidates have been developed whereby alternative hydrogen-bonding patterns and hydrophobic and packing interactions have turned out to be the most promising base-pairing concepts to date. The key in many applications is the highly efficient and selective acceptance of artificial base pairs by DNA polymerases, which enables amplification of the modified DNA. In this Review, computational as well as exptl. studies that were performed to characterize the pairing behavior of UBPs in free duplex DNA or bound to the active site of KlenTaq DNA polymerase are highlighted. The structural studies, on the one hand, elucidate how base pairs lacking hydrogen bonds are accepted by these enzymes and, on the other hand, highlight the influence of one or several consecutive UBPs on the structure of a DNA double helix. Understanding these concepts facilitates optimization of future UBPs for the manifold fields of applications.
- 16Li, L.; Degardin, M.; Lavergne, T.; Malyshev, D. A.; Dhami, K.; Ordoukhanian, P.; Romesberg, F. E. Natural-like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applications. J. Am. Chem. Soc. 2014, 136, 826– 829, DOI: 10.1021/ja408814gGoogle Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1KktLnO&md5=5d290f34aab307b9a965dfca3386dcf7Natural-like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applicationsLi, Lingjun; Degardin, Melissa; Lavergne, Thomas; Malyshev, Denis A.; Dhami, Kirandeep; Ordoukhanian, Phillip; Romesberg, Floyd E.Journal of the American Chemical Society (2014), 136 (3), 826-829CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors synthesized a panel of unnatural base pairs whose pairing depends on hydrophobic and packing forces and identify dTPT3 (I)-dNaM, which is PCR amplified with a natural base pair-like efficiency and fidelity. In addn., the I scaffold is uniquely tolerant of attaching a propargyl amine linker, resulting in the dTPT3PA-dNaM pair, which is amplified only slightly less well. The identification of I represents significant progress toward developing an unnatural base pair for the in vivo expansion of an organism's genetic alphabet and for a variety of in vitro biotechnol. applications where it is used to site-specifically label amplified DNA, and it also demonstrates for the first time that hydrophobic and packing forces are sufficient to mediate natural-like replication.
- 17Zhang, Y.; Ptacin, J. L.; Fischer, E. C.; Aerni, H. R.; Caffaro, C. E.; San Jose, K.; Feldman, A. W.; Turner, C. R.; Romesberg, F. E. A semi-synthetic organism that stores and retrieves increased genetic information. Nature 2017, 551, 644– 647, DOI: 10.1038/nature24659Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWhtL7L&md5=d04861904783b1a852d4089b28f09acfA semi-synthetic organism that stores and retrieves increased genetic informationZhang, Yorke; Ptacin, Jerod L.; Fischer, Emil C.; Aerni, Hans R.; Caffaro, Carolina E.; San Jose, Kristine; Feldman, Aaron W.; Turner, Court R.; Romesberg, Floyd E.Nature (London, United Kingdom) (2017), 551 (7682), 644-647CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Since at least the last common ancestor of all life on Earth, genetic information has been stored in a four-letter alphabet that is propagated and retrieved by the formation of two base pairs. The central goal of synthetic biol. is to create new life forms and functions, and the most general route to this goal is the creation of semi-synthetic organisms whose DNA harbors two addnl. letters that form a third, unnatural base pair. Previous efforts to generate such semi-synthetic organisms culminated in the creation of a strain of Escherichia coli that, by virtue of a nucleoside triphosphate transporter from Phaeodactylum tricornutum, imports the requisite unnatural triphosphates from its medium and then uses them to replicate a plasmid contg. the unnatural base pair dNaM-dTPT3. Although the semi-synthetic organism stores increased information when compared to natural organisms, retrieval of the information requires in vivo transcription of the unnatural base pair into mRNA and tRNA, aminoacylation of the tRNA with a non-canonical amino acid, and efficient participation of the unnatural base pair in decoding at the ribosome. Here we report the in vivo transcription of DNA contg. dNaM and dTPT3 into mRNAs with two different unnatural codons and tRNAs with cognate unnatural anticodons, and their efficient decoding at the ribosome to direct the site-specific incorporation of natural or non-canonical amino acids into superfolder green fluorescent protein. The results demonstrate that interactions other than hydrogen bonding can contribute to every step of information storage and retrieval. The resulting semi-synthetic organism both encodes and retrieves increased information and should serve as a platform for the creation of new life forms and functions.
- 18Kimoto, M.; Kawai, R.; Mitsui, T.; Yokoyama, S.; Hirao, I. An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules. Nucleic Acids Res. 2009, 37, e14 DOI: 10.1093/nar/gkn956Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlGksL8%253D&md5=8812c6b48c1a2c611efdd4f77afda742An unnatural base pair system for efficient PCR amplification and functionalization of DNA moleculesKimoto, Michiko; Kawai, Rie; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, IchiroNucleic Acids Research (2009), 37 (2), e14/1-e14/9CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates contg. unnatural bases, natural and unnatural base triphosphates, and a 3'→5' exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library contg. the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) contg. the unnatural base pair can be amplified 107-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA mols. from mixts. with excess amts. (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.
- 19Yang, Z.; Sismour, A. M.; Sheng, P.; Puskar, N. L.; Benner, S. A. Enzymatic incorporation of a third nucleobase pair. Nucleic Acids Res. 2007, 35, 4238– 4249, DOI: 10.1093/nar/gkm395Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXos12nsr4%253D&md5=9b802c5f84a6e199ced685bb6fa31d72Enzymatic incorporation of a third nucleobase pairYang, Zunyi; Sismour, A. Michael; Sheng, Pinpin; Puskar, Nyssa L.; Benner, Steven A.Nucleic Acids Research (2007), 35 (13), 4238-4249CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)DNA polymerases are identified that copy a non-std. nucleotide pair joined by a hydrogen bonding pattern different from the patterns joining the dA:T and dG:dC pairs. 6-Amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) implements the non-std. 'small' donor-donor-acceptor (pyDDA) hydrogen bonding pattern. 2-Amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP) implements the 'large' acceptor-acceptor-donor (puAAD) pattern. These nucleobases were designed to present electron d. to the minor groove, d. hypothesized to help det. specificity for polymerases. Consistent with this hypothesis, both dZTP and dPTP are accepted by many polymerases from both Families A and B. Further, the dZ:dP pair participates in PCR reactions catalyzed by Taq, Vent (exo-) and Deep Vent (exo-) polymerases, with 94.4%, 97.5% and 97.5%, resp., retention per round. The dZ:dP pair appears to be lost principally via transition to a dC:dG pair. This is consistent with a mechanistic hypothesis that deprotonated dZ (presenting a pyDAA pattern) complements dG (presenting a puADD pattern), while protonated dC (presenting a pyDDA pattern) complements dP (presenting a puAAD pattern). This hypothesis, grounded in the Watson-Crick model for nucleobase pairing, was confirmed by studies of the pH-dependence of mismatching. The dZ:dP pair and these polymerases, should be useful in dynamic architectures for sequencing, mol.-, systems- and synthetic-biol.
- 20Yang, Z.; Chen, F.; Alvarado, J. B.; Benner, S. A. Amplification, mutation, and sequencing of a six-letter synthetic genetic system. J. Am. Chem. Soc. 2011, 133, 15105– 15112, DOI: 10.1021/ja204910nGoogle Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFaks7zL&md5=cc4561e9527ca359c82494deb9e0be2dAmplification, mutation, and sequencing of a six-letter synthetic genetic systemYang, Zun-Yi; Chen, Fei; Alvarado, J. Brian; Benner, Steven A.Journal of the American Chemical Society (2011), 133 (38), 15105-15112CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The next goals in the development of a synthetic biol. that uses artificial genetic systems will require chem.-biol. combinations that allow the amplification of DNA contg. any no. of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technol. is needed to sequence artificial genetic DNA mols. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four std. nucleotides (G, A, C, and T) and two addnl. nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theor. cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.
- 21Ashwood, B.; Pollum, M.; Crespo-Hernández, C. E. Can a Six-Letter Alphabet Increase the Likelihood of Photochemical Assault to the Genetic Code?. Chem. – Eur. J. 2016, 22, 16648– 16656, DOI: 10.1002/chem.201602160Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1ClsLvM&md5=cc289085cffc83044b2879a95b5f4456Can a Six-Letter Alphabet Increase the Likelihood of Photochemical Assault to the Genetic Code?Ashwood, Brennan; Pollum, Marvin; Crespo-Hernandez, Carlos E.Chemistry - A European Journal (2016), 22 (46), 16648-16656CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)In 2014, two unnatural nucleosides, d5SICS and dNaM, were shown to selectively base pair and replicate with high fidelity in a modified strain of E. coli, thus effectively expanding its genetic alphabet from four to six letters. More recently, a significant redn. in cell proliferation was reported in cells cultured with d5SICS, and putatively with dNaM, upon exposure to brief periods of near-visible radiation. The photosensitizing properties of the lowest-energy excited triplet state of both d5SICS and dNaM were implicated in their cytotoxicity. Importantly, however, the excited-state mechanisms by which near-visible excitation populates the triplet states of d5SICS and dNaM are currently unknown. In this study, steady-state and time-resolved spectroscopies are combined with quantum-chem. calcns. in order to reveal the excited-state relaxation mechanisms leading to efficient population of the triplet states in these unnatural nucleosides in soln. It is shown that excitation of d5SICS or dNaM with near-visible light leads overwhelmingly to ultrafast population of their triplet states on the femtosecond time scale. The results presented in this work lend strong support to the proposal that photoexcitation of these unnatural nucleosides can accelerate oxidatively generated damage to DNA and other biomols. within the cellular environment.
- 22Bhattacharyya, K.; Datta, A. Visible-Light-Mediated Excited State Relaxation in Semi-Synthetic Genetic Alphabet: d5SICS and dNaM. Chem. – Eur. J. 2017, 23, 11494– 11498, DOI: 10.1002/chem.201702583Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1KqurbJ&md5=0ad08a3d291ccb9a7a9d23ea7091720bVisible-light-mediated excited state relaxation in semi-synthetic genetic alphabet: d5SICS and dNaMBhattacharyya, Kalishankar; Datta, AyanChemistry - A European Journal (2017), 23 (48), 11494-11498CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)The excited state dynamics of an unnatural base pair (UBP) d5SICS/dNaM were investigated by accurate ab-initio calcns. Time-dependent d. functional and high-level multireference calcns. (MS-CASPT2) were performed to elucidate the excitation of this UBP and its excited state relaxation mechanism. After excitation to the bright state S2 (ππ*), it decays to the S1 state and then undergoes efficient intersystem crossing to the triplet manifold. The presence of sulfur atom in d5SICS leads to strong spin-orbit coupling (SOC) and a small energy gap that facilitates intersystem crossing from S1 (nsπ*) to T2 (ππ*) followed by internal conversion to T1 state. Similarly in dNaM, the deactivation pathway follows analogous trends. CASPT2 calcns. suggest that the S1 (ππ*) state is a dark state below the accessible S2 (ππ*) bright state. During the ultrafast deactivation, it exhibits bond length inversion. From S1 state, significant SOC leads the population transfer to T3 due to a smaller energy gap. Henceforth, fast internal conversion occurs from T3 to T2 followed by T1. From time-dependent trajectory surface hopping dynamics, it is found that excited state relaxation occurs on a sub-picosecond timescale in d5SICS and dNaM. Our findings strongly suggest that there is enough energy available in triplet state of UBP to generate reactive oxygen species and induce phototoxicity with respect to cellular DNA.
- 23Pollum, M.; Ashwood, B.; Jockusch, S.; Lam, M.; Crespo-Hernández, C. E. Unintended Consequences of Expanding the Genetic Alphabet. J. Am. Chem. Soc. 2016, 138, 11457– 11460, DOI: 10.1021/jacs.6b06822Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVWlsbbM&md5=73a69f7e204452afb85b39c530d95aecUnintended Consequences of Expanding the Genetic AlphabetPollum, Marvin; Ashwood, Brennan; Jockusch, Steffen; Lam, Minh; Crespo-Hernandez, Carlos E.Journal of the American Chemical Society (2016), 138 (36), 11457-11460CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The base pair d5SICS·dNaM was recently reported to incorporate and replicate in the DNA of a modified strain of Escherichia coli, thus making the world's first stable semisynthetic organism. This newly expanded genetic alphabet may allow organisms to store considerably more information in order to translate proteins with unprecedented enzymic activities. Importantly, however, there is currently no knowledge of the photochem. properties of d5SICS or dNaM-properties that are central to the chem. integrity of cellular DNA. In this contribution, it is shown that excitation of d5SICS or dNaM with near-visible light leads to efficient trapping of population in the nucleoside's excited triplet state in high yield. Photoactivation of these long-lived, reactive states is shown to photosensitize cells, leading to the generation of reactive oxygen species and to a marked decrease in cell proliferation, thus warning scientists of the potential phototoxic side effects of expanding the genetic alphabet.
- 24Feldmann, A. W.; Romesberg, F. E. Expansion of the genetic alphabet: A Chemist’s approach to synthetic biology. Acc. Chem. Res. 2018, 51, 394– 403, DOI: 10.1021/acs.accounts.7b00403Google ScholarThere is no corresponding record for this reference.
- 25Lavergne, T.; Malyshev, D. A.; Romesberg, F. E. Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairs. Chem. – Eur. J. 2012, 18, 1231– 1239, DOI: 10.1002/chem.201102066Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhs1Ghur3L&md5=e3fe084c25ab1596e07e721de72d4408Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairsLavergne, Thomas; Malyshev, Denis A.; Romesberg, Floyd E.Chemistry - A European Journal (2012), 18 (4), 1231-1239, S1231/1-S1231/27CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)Expansion of the genetic alphabet with an unnatural base pair is a long-standing goal of synthetic biol. The authors have developed a class of unnatural base pairs, formed between d5SICS and analogs of dMMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, the authors report the synthesis of five new dMMO2 analogs bearing different substituents designed to be oriented into the developing major groove and an anal. of their insertion opposite d5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the anal. of the previously optimized pair, dNaM-d5SICS, to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examd. The resulting structure-activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addn., the authors identify an analog (dNMO1) that is a better partner for d5SICS than any of the previously identified dMMO2 analogs with the exception of dNaM. They also found that dNaM-d5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.
- 26Dhami, K.; Malyshev, D. A.; Ordoukhanian, P.; Kubelka, T.; Hocek, M.; Romesberg, F. E. Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabet. Nucleic Acids Res. 2014, 42, 10235– 10244, DOI: 10.1093/nar/gku715Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVCmtLnL&md5=4eefc0a675b6075fd06398d74400ad19Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabetDhami, Kirandeep; Malyshev, Denis A.; Ordoukhanian, Phillip; Kubelka, Tomas; Hocek, Michal; Romesberg, Floyd E.Nucleic Acids Research (2014), 42 (16), 10235-10244CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)We have developed a family of unnatural base pairs (UBPs), which rely on hydrophobic and packing interactions for pairing and which are well replicated and transcribed. While the pair formed between d5SICS and dNaM (d5SICS-dNaM) has received the most attention, and has been used to expand the genetic alphabet of a living organism, recent efforts have identified dTPT3-dNaM, which is replicated with even higher fidelity. These efforts also resulted in more UBPs than could be independently analyzed, and thus we now report a PCR-based screen to identify the most promising. While we found that dTPT3-dNaM is generally the most promising UBP, we identified several others that are replicated nearly as well and significantly better than d5SICS-dNaM, and are thus viable candidates for the expansion of the genetic alphabet of a living organism. Moreover, the results suggest that continued optimization should be possible, and that the putatively essential hydrogen-bond acceptor at the position ortho to the glycosidic linkage may not be required. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new structure-activity relationship data and importantly identify multiple new candidates for in vivo evaluation and further optimization.
- 27Lavergne, T.; Degardin, M.; Malyshev, D. A.; Quach, H. T.; Dhami, K.; Ordoukhanian, P.; Romesberg, F. E. Expanding the scope of replicable unnatural DNA: stepwise optimization of a predominantly hydrophobic base pair. J. Am. Chem. Soc. 2013, 135, 5408– 5419, DOI: 10.1021/ja312148qGoogle Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltV2msbY%253D&md5=0aa95cf5b916e8b619497821d808fe84Expanding the Scope of Replicable Unnatural DNA: Stepwise Optimization of a Predominantly Hydrophobic Base PairLavergne, Thomas; Degardin, Melissa; Malyshev, Denis A.; Quach, Henry T.; Dhami, Kirandeep; Ordoukhanian, Phillip; Romesberg, Floyd E.Journal of the American Chemical Society (2013), 135 (14), 5408-5419CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)As part of an ongoing effort to expand the genetic alphabet for in vitro and eventually in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs exemplified by d5SICS-dMMO2 and d5SICS-dNaM. When incorporated into DNA, the latter is replicated and transcribed with greater efficiency and fidelity than the former; however, previous optimization efforts identified the para and methoxy-distal meta positions of dMMO2 as particularly promising for further optimization. Here, we report the stepwise optimization of dMMO2 via the synthesis and evaluation of 18 novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most promising analogs with meta substituents. Subject to size constraints, we find that para substituents can optimize replication via both steric and electronic effects and that meta methoxy groups are unfavorable, while fluoro substituents can be beneficial or deleterious depending on the para substituent. In addn., we find that improvements in the efficiency of unnatural triphosphate insertion translate most directly into higher fidelity replication. Importantly, we identify multiple, unique base pair derivs. that when incorporated into DNA are well replicated. The most promising, d5SICS-dFEMO, is replicated under some conditions with greater efficiency and fidelity than d5SICS-dNaM. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new SAR data, and importantly identify multiple new candidates for eventual in vivo evaluation.
- 28Pollum, M.; Crespo-Hernández, C. E. Communication: the dark singlet state as a doorway state in the ultrafast and efficient intersystem crossing dynamics in 2-thiothymine and 2-thiouracil. J. Chem. Phys. 2014, 140, 071101 DOI: 10.1063/1.4866447Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjslKmt7w%253D&md5=666eb3d15e330fb467ad89e97b939214Communication: The dark singlet state as a doorway state in the ultrafast and efficient intersystem crossing dynamics in 2-thiothymine and 2-thiouracilPollum, Marvin; Crespo-Hernandez, Carlos E.Journal of Chemical Physics (2014), 140 (7), 071101/1-071101/5CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)Femtosecond broadband transient absorption expts. are reported for 2-thiothymine and 2-thiouracil in aq. buffer soln. and in acetonitrile. The S1(nπ*) state acts as a doorway state in the ultrafast and efficient population of the T1(ππ*) state upon 316 nm excitation. A sequential kinetic model is presented to explain the excited-state dynamics in 2-thiothymine and 2-thiouracil upon UVA excitation: S2(ππ*) → S1(nπ*) → T1(ππ*). The exptl. results are also used to scrutinize the excited-state relaxation pathways recently predicted for 2-thiouracil at the CASPT2//CASSCF level of theory. The efficient population of the T1(ππ*) state for both 2-thiothymine and 2-thiouracil in a few hundreds of femtoseconds lends further support to the emerging idea that thiobase derivs. exhibit photo-toxic properties that can be effectively harnessed in photo-chemotherapeutic applications. (c) 2014 American Institute of Physics.
- 29Pollum, M.; Jockusch, S.; Crespo-Hernández, C. E. 2,4-Dithiothymine as a potent UVA chemotherapeutic agent. J. Am. Chem. Soc. 2014, 136, 17930– 17933, DOI: 10.1021/ja510611jGoogle Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitFOgsLzO&md5=a8d7ac77d8ff134caf66b1d7df63671a2,4-Dithiothymine as a Potent UVA Chemotherapeutic AgentPollum, Marvin; Jockusch, Steffen; Crespo-Hernandez, Carlos E.Journal of the American Chemical Society (2014), 136 (52), 17930-17933CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Substitution of both oxygen atoms in the exocyclic carbonyl groups of the thymine chromophore by sulfur atoms results in a remarkable red shift of its absorption spectrum from an absorption max. at 267 nm in thymidine to 363 nm in 2,4-dithiothymine (ΔE = 9905 cm-1). A single sulfur substitution of a carbonyl group in the thymine chromophore at position 2 or 4 results in a significantly smaller red shift in the absorption max., which depends sensitively on the position at which the sulfur atom is substituted, varying from 275 nm in 2-thiothymine to 335 nm in 4-thiothymidine. Femtosecond transient absorption spectroscopy reveals that excitation of 2,4-dithiothymine at 335 or 360 nm leads to the ultrafast population of the triplet state, with an intersystem crossing lifetime of 180 ± 40 fs-the shortest intersystem crossing lifetime of any DNA base deriv. studied so far in aq. soln. Surprisingly, the degree and position at which the sulfur atom is substituted have important effects on the magnitude of the intersystem crossing rate const., showing a 1.2-, 3.2-, and 4.2-fold rate increases for 2-thiothymine, 4-thiothymidine, and 2,4-dithiothymine, resp., relative to that of thymidine, whereas the triplet yield increases 60-fold to near unity, independent of the site of sulfur atom substitution. While the natural thymine monomers owe their high degree of photostability to ultrafast internal conversion to the ground state and low triplet yields, the near-unity triplet yields in the thiothymine series account for their potent photosensitization properties. Nanosecond time-resolved luminescence spectroscopy shows that 4-thiothymidine and 2,4-dithiothymine are efficient singlet oxygen generators, with singlet oxygen quantum yields of 0.42 ± 0.02 and 0.46 ± 0.02, resp., in O2-satd. acetonitrile soln. Taken together, these photophys. measurements strongly suggest that 2,4-dithiothymine can act as a more effective UVA chemotherapeutic agent than the currently used 4-thiothymidine, esp. in deeper-tissue chemotherapeutic applications.
- 30Pollum, M.; Jockusch, S.; Crespo-Hernández, C. E. Increase in the photoreactivity of uracil derivatives by doubling thionation. Phys. Chem. Chem. Phys. 2015, 17, 27851– 27861, DOI: 10.1039/C5CP04822BGoogle Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFKhtbjN&md5=9931a8877b0f1e9b31fc665c6fb9ec3bIncrease in the photoreactivity of uracil derivatives by doubling thionationPollum, M.; Jockusch, S.; Crespo-Hernandez, C. E.Physical Chemistry Chemical Physics (2015), 17 (41), 27851-27861CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)The ability of 4-thiouracil to strongly absorb UVA radiation and to populate a reactive triplet state in high yield has enabled its use as a versatile photocrosslinker for nearly 50 years. In this contribution, we present a detailed spectroscopic and photochem. investigation of the 2-thiouracil, 4-thiouracil, and 2,4-dithiouracil series in an effort to further advance this chem. and to scrutinize the photoreactivity of 2,4-dithiouracil. Our results reveal that excitation of 2,4-dithiouracil leads to intersystem crossing to the triplet manifold in 220 ± 40 fs, which enables the population of the reactive triplet state with near unity yield (ΦT = 0.90 ± 0.15) and ultimately leads to a ca. 50% singlet oxygen generation (ΦΔ = 0.49 ± 0.02)-one of the highest singlet oxygen yields reported to date for a photoexcited thiobase. In addn., the long-lived triplet state of 2,4-dithiouracil reacts efficiently with the nucleic acid base adenine 5'-monophosphate through a direct, oxygen-independent photocycloaddn. mechanism and at a rate that is at least 3-fold faster than that of 4-thiouracil under equal conditions. The new physico-chem. insights reported for these RNA-thiobase derivs. are compared to those of the DNA and RNA bases and the DNA-thiobase derivs. Furthermore, the strong near-visible absorption and increased photoreactivity measured for 2,4-dithiouracil lays a solid foundation for developing RNA-targeted photocrosslinking and phototherapeutic agents that are more effective than those currently available.
- 31Dien, V. T.; Holcomb, M.; Feldman, A. W.; Fischer, E. C.; Dwyer, T. J.; Romesberg, F. E. Progress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic Alphabet. J. Am. Chem. Soc. 2018, 140, 16115– 16123, DOI: 10.1021/jacs.8b08416Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFGmtrbP&md5=206c2634fd4753e9bdaafd9959f3023bProgress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic AlphabetDien, Vivian T.; Holcomb, Matthew; Feldman, Aaron W.; Fischer, Emil C.; Dwyer, Tammy J.; Romesberg, Floyd E.Journal of the American Chemical Society (2018), 140 (47), 16115-16123CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have developed a family of unnatural base pairs (UBPs), exemplified by the pair formed between dNaM and dTPT3, for which pairing is mediated not by complementary hydrogen bonding but by hydrophobic and packing forces. These UBPs enabled the creation of the first semisynthetic organisms (SSOs) that store increased genetic information and use it to produce proteins contg. noncanonical amino acids. However, retention of the UBPs was poor in some sequence contexts. Here, to optimize the SSO, we synthesize two novel benzothiophene-based dNaM analogs, dPTMO and dMTMO, and characterize the corresponding UBPs, dPTMO-dTPT3 and dMTMO-dTPT3. We demonstrate that these UBPs perform similarly to, or slightly worse than, dNaM-dTPT3in vitro. However, in the in vivo environment of an SSO, retention of dMTMO-dTPT3, and esp. dPTMO-dTPT3, is significantly higher than that of dNaM-dTPT3. This more optimal in vivo retention results from better replication, as opposed to more efficient import of the requisite unnatural nucleoside triphosphates. Modeling studies suggest that the more optimal replication results from specific internucleobase interactions mediated by the thiophene sulfur atoms. Finally, we show that dMTMO and dPTMO efficiently template the transcription of RNA contg. TPT3 and that their improved retention in DNA results in more efficient prodn. of proteins with noncanonical amino acids. This is the first instance of using performance within the SSO as part of the UBP evaluation and optimization process. From a general perspective, the results demonstrate the importance of evaluating synthetic biol. "parts" in their in vivo context and further demonstrate the ability of hydrophobic and packing interactions to replace the complementary hydrogen bonding that underlies the replication of natural base pairs. From a more practical perspective, the identification of dMTMO-dTPT3 and esp. dPTMO-dTPT3 represents significant progress toward the development of SSOs with an unrestricted ability to store and retrieve increased information.
- 32Jahiruddin, S.; Mandal, N.; Datta, A. Structure and Electronic Properties of Unnatural Base Pairs: The Role of Dispersion Interactions. ChemPhysChem 2018, 19, 67– 74, DOI: 10.1002/cphc.201700997Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFGmtbjE&md5=e5af77161a0f2967c67bf044b889a9d9Structure and electronic properties of unnatural base pairs: The role of dispersion interactionsJahiruddin, Sk; Mandal, Nilangshu; Datta, AyanChemPhysChem (2018), 19 (1), 67-74CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)Recent reports of the successful incorporation of unnatural base pairs (UBPs), such as d5SICS-dNaM, in the gene sequence and replication with DNA is an important milestone in synthetic biol. Followed by this, several other UBPs, such as dTPT3-dNaM, dTPT3-dFIMO, dTPT3-IMO, dTPT3-FEMO, FTPT3-NaM, FTPT3-FIMO, FTPT3-IMO, and FTPT3-FEMO, have demonstrated similar or better retention and fidelity inside cells. Of these base pairs, dNaM-dTPT3 has been optimized to be a better fit inside a pAIO plasmid. Based on both implicit and explicit dispersion-cor. d. functional theory (DFT) calcns., we show that although this set of UBPs is significantly diverse in elemental and structural configuration, the members do share a common trait of favoring a slipped parallel stacked dimer arrangement. Unlike the natural bases (A, T, G, C, and U), this set of UBPs has a negligible affinity for a Watson-Crick (WC)-type planar structure because they are invariably more stable within slipped parallel stacked orientations. We also obsd. that all the UBPs have either similar or higher binding energies with the natural bases in similar stacked orientations. When arranged between two natural base pairs, the UBPs exhibited a binding energy similar to that of three-base sequences of natural bases. Our computational data show that the most promising base pairs are 5SICS-NaM, TPT3-NaM, and TPT3-FEMO. These results are consistent with recent progress on exptl. research into UBPs along with our previous calcns. on the d5SICS-dNaM pair and, therefore, strengthen the hypothesis that hydrogen bonding might not be absolutely essential and that interbase stacking dispersion interactions play a key role in the stabilization of genetic materials.
- 33Negi, I.; Kathuria, P.; Sharma, P.; Wetmore, S. D. How do hydrophobic nucleobases differ from natural DNA nucleobases? Comparison of structural features and duplex properties from QM calculations and MD simulations. Phys. Chem. Chem. Phys. 2017, 19, 16365– 16374, DOI: 10.1039/C7CP02576AGoogle Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpsVCmt7k%253D&md5=bdd66a0051cfd743e7cec50b48c75744How do hydrophobic nucleobases differ from natural DNA nucleobases? Comparison of structural features and duplex properties from QM calculations and MD simulationsNegi, Indu; Kathuria, Preetleen; Sharma, Purshotam; Wetmore, Stacey D.Physical Chemistry Chemical Physics (2017), 19 (25), 16365-16374CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)Computational (DFT and MD simulation) methods are employed to systematically characterize the structural and energetic properties of five hydrophobic nucleobases (FEMO, MMO2, NaM, 5SICS and TPT3) that constitute four unnatural base pairs (FEMO:5SICS, MMO2:5SICS, NaM:5SICS and TPT3:NaM). These hydrophobic bases have been recently shown to be replicated when present between natural bases in DNA duplexes, with the highest replication fidelity and efficiency occurring for the TPT3:NaM pair. Our QM calcns. suggest that the preferred (anti) glycosidic orientations of nucleosides contg. hydrophobic bases are similar to the natural DNA nucleosides despite differences in their chem. structures. However, due to the inability to form interbase hydrogen bonds, hydrophobic base pairs intrinsically prefer nonplanar, distorted geometries, many of which are stabilized through π-π stacking interactions. Furthermore, the intrinsic stacking potential between a hydrophobic and a natural base is similar to that between two natural bases, indicating that the strength of stacking interactions in DNA duplexes contg. hydrophobic bases is likely comparable to natural DNA. However, in contrast to the isolated base-pair geometries, our MD simulations suggest that the hydrophobic base pairs adopt variable geometries within DNA, which range from stacked (5SICS:FEMO) to nearly planar (5SICS:NaM and SICS:MMO2) to planar (TPT3:NaM). As a result, the duplex structural features at the site of modification depend on the identity of the hydrophobic base pair, where the TPT3:NaM pair causes the least structural changes compared to natural DNA. Overall, the structural insight obtained from our calcns. on DNA contg. hydrophobic base pairs explains the exptl.-obsd. higher fidelity and efficiency during replication of TPT3:NaM compared to other hydrophobic nucleobase pairs. By providing valuable structural information that explains the intrinsic and duplex properties of this class of unnatural nucleobases, the present work may aid the future design of improved hydrophobic analogs.
- 34Leconte, A. M.; Hwang, G. T.; Matsuda, S.; Capek, P.; Hari, Y.; Romesberg, F. E. Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet. J. Am. Chem. Soc. 2008, 130, 2336– 2343, DOI: 10.1021/ja078223dGoogle Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXps1KmtQ%253D%253D&md5=e8a92b3f3735fd4a116ca3a69534485bDiscovery, Characterization, and Optimization of an Unnatural Base Pair for Expansion of the Genetic AlphabetLeconte, Aaron M.; Hwang, Gil Tae; Matsuda, Shigeo; Capek, Petr; Hari, Yoshiyuki; Romesberg, Floyd E.Journal of the American Chemical Society (2008), 130 (7), 2336-2343CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)DNA is inherently limited by its four natural nucleotides. Efforts to expand the genetic alphabet, by addn. of an unnatural base pair, promise to expand the biotechnol. applications available for DNA as well as to be an essential first step toward expansion of the genetic code. We have conducted two independent screens of hydrophobic unnatural nucleotides to identify novel candidate base pairs that are well recognized by a natural DNA polymerase. From a pool of 3600 candidate base pairs, both screens identified the same base pair, dSICS:dMMO2, which we report here. Using a series of related analogs, we performed a detailed structure-activity relationship anal., which allowed us to identify the essential functional groups on each nucleobase. From the results of these studies, we designed an optimized base pair, d5SICS:dMMO2, which is efficiently and selectively synthesized by Kf within the context of natural DNA.
- 35Henry, A. A.; Yu, C.; Romesberg, F. E. Determinants of unnatural nucleobase stability and polymerase recognition. J. Am. Chem. Soc. 2003, 125, 9638– 9646, DOI: 10.1021/ja035398oGoogle Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXlsFOhsb4%253D&md5=f1e6a734e700d316fab4e25b738666f4Determinants of Unnatural Nucleobase Stability and Polymerase RecognitionHenry, Allison A.; Yu, Chengzhi; Romesberg, Floyd E.Journal of the American Chemical Society (2003), 125 (32), 9638-9646CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Six new unnatural nucleobases have been synthesized and characterized in terms of stability and selectivity of self-pairing in duplex DNA and efficiency and fidelity of self-pairing during polymerase-mediated replication. Each nucleobase has a conserved ring structure but differs from the others in its specific pattern of substitution with oxygen and sulfur atoms. Heteroatom derivatization within the conserved scaffold is shown to have only moderate effects on unnatural self-pair synthesis by the polymerase; larger effects were obsd. on the thermal stability and polymerase-mediated extension of the self-pairs. The largest effects of heteroatom substitution were on the stability and synthesis of mispairs between the unnatural and natural bases. Certain heteroatom substitutions were found to have a general effect while others were found to have effects that were specific for a particular unnatural or natural base. The data are useful for designing stable and replicable third base pairs and for understanding the contributions of nucleobase shape, polarity, and polarizability to the stability and replication of DNA.
- 36New, J. S.; Christopher, W. L.; Yevich, J. P.; Butler, R.; Schlemmer, R. F., Jr.; VanderMaelen, C. P.; Cipollina, J. A. The thieno[3,2-c]pyridine and furo[3,2-c]pyridine rings: new pharmacophores with potential antipsychotic activity. J. Med. Chem. 1989, 32, 1147– 1156, DOI: 10.1021/jm00126a002Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXitlWmurw%253D&md5=098cfbe9523216238b07e82c1e01f0c4The thieno[3,2-c]pyridine and furo[3,2-c]pyridine rings: new pharmacophores with potential antipsychotic activityNew, James S.; Christopher, William L.; Yevich, Joseph P.; Butler, Rhett; Schlemmer, R. Francis, Jr.; VanderMaelen, Cam P.; Cipollina, Joseph A.Journal of Medicinal Chemistry (1989), 32 (6), 1147-56CODEN: JMCMAR; ISSN:0022-2623.Imidobutylpiperazinylpyrrolo-, -thieno-, and -furo[3,2-c]pyridines (e.g. I, X = O, Me2C; X1 = MeN, S, O) were synthesized. A no. of I (X1 = S, O) had significant activity in the blockade of apomorphine stereotypy and apomorphine-induced climbing, the Sidman avoidance response, and the conditioned avoidance response. In addn., while potent affinity for serotonin 5-HT2 and 5-HT2 receptors was obsd. for both the thieno- and furo[3,2-c]pyridine derivs., the interaction of these mols. with the dopamine D2 receptor was weak. Electrophysiol. studies of the lead prototypes I (X = Me2C, X1 = S; X = X1 = O), indicate these two mols. have distinctively different effects on dopamine neurons in areas A9 and A10. Despite the similarity these mols. share in their behavioral indexes of antipyschotic activity, it is likely that the thieno- and furo[3,2-c]pyridine rings employ different mechanisms to achieve this convergence of biol. effects.
- 37Asagarasu, A.; Matsui, T.; Hayashi, H.; Tamaoki, S.; Yamauchi, Y.; Sato, M. Design and Synthesis of Piperazinylpyridine Derivatives as Novel 5-HT1A Agonists/5-HT3 Antagonists for the Treatment of Irritable Bowel Syndrome (IBS). Chem. Pharm. Bull. 2009, 57, 34– 42, DOI: 10.1248/cpb.57.34Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmsVGltr8%253D&md5=f075d0d527feed2dfd129199deb3d775Design and synthesis of piperazinylpyridine derivatives as novel 5-HT1A agonists/5-HT3 antagonists for the treatment of irritable bowel syndrome (IBS)Asagarasu, Akira; Matsui, Teruaki; Hayashi, Hiroyuki; Tamaoki, Satoru; Yamauchi, Yukinao; Sato, MichitakaChemical & Pharmaceutical Bulletin (2009), 57 (1), 34-42CODEN: CPBTAL; ISSN:0009-2363. (Pharmaceutical Society of Japan)We have prepd. a series of piperazinylpyridine derivs. for the treatment of irritable bowel syndrome (IBS). These compds., which were designed by pharmacophore anal., bind to both serotonin subtype 1A (5-HT1A) and subtype 3 (5-HT3) receptors. The nitrogen atom of the isoquinoline, a methoxy group and piperazine were essential to the pharmacophore for binding to these receptors. We also synthesized furo- and thienopyridine derivs. according to structure-activity relationship analyses. Compd. 17a (TZB-20810, I) had high affinities to these receptors and exhibited 5-HT1A agonistic activity and 5-HT3 antagonistic activity concurrently, and is a promising drug for further development in the treatment of IBS.
- 38Gueymard, C. A.; Myers, D.; Emery, K. Proposed reference irradiance spectra for solar energy systems testing. Solar Energy. 2002, 73, 443– 467, DOI: 10.1016/S0038-092X(03)00005-7Google ScholarThere is no corresponding record for this reference.
- 39Morris, S. E.; Feldman, A. W.; Romesberg, F. E. Synthetic Biology Parts for the Storage of Increased Genetic Information in Cells. ACS Synth. Biol. 2017, 6, 1834– 1840, DOI: 10.1021/acssynbio.7b00115Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFyhu7vL&md5=4ec70cf80be8c5412e48b4b72f5a464aSynthetic biology parts for the storage of increased genetic information in cellsMorris, Sydney E.; Feldman, Aaron W.; Romesberg, Floyd E.ACS Synthetic Biology (2017), 6 (10), 1834-1840CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)To bestow cells with novel forms and functions, the goal of synthetic biol., we have developed the unnatural nucleoside triphosphates dNaMTP and dTPT3TP, which form an unnatural base pair (UBP) and expand the genetic alphabet. While the UBP may be retained in the DNA of a living cell, its retention is sequence-dependent. We now report a steady-state kinetic characterization of the rate with which the Klenow fragment of E. coli DNA polymerase I synthesizes the UBP and its mispairs in a variety of sequence contexts. Correct UBP synthesis is as efficient as for a natural base pair, except in one sequence context, and in vitro performance is correlated with in vivo performance. The data elucidate the determinants of efficient UBP synthesis, show that the dNaM-dTPT3 UBP is the first generally recognized natural-like base pair, and importantly, demonstrate that dNaMTP and dTPT3TP are well optimized and standardized parts for the expansion of the genetic alphabet.
- 40Ouaray, Z.; Benner, S. A.; Georgiadis, M. M.; Richards, N. G. J. Building better polymerases: Engineering the replication of expanded genetic alphabets. J. Biol. Chem. 2020, 295, 17046– 17059, DOI: 10.1074/jbc.REV120.013745Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXpvVWisw%253D%253D&md5=c27c40b83d9428d8e4898a9ebf880707Building better polymerases: engineering the replication of expanded genetic alphabetsOuaray, Zahra; Benner, Steven A.; Georgiadis, Millie M.; Richards, Nigel G. J.Journal of Biological Chemistry (2020), 295 (50), 17046-17059CODEN: JBCHA3; ISSN:1083-351X. (American Society for Biochemistry and Molecular Biology)A review. DNA polymerases are today used throughout scientific research, biotechnol., and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here esp., natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chem. and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymic, and mol. dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.
- 41Percze, K.; Meszaros, T. Analysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA Polymerases. ChemBioChem 2020, 21, 2939– 2944, DOI: 10.1002/cbic.202000236Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlOju7rO&md5=d868026ed6003b8049e79ca55011cbdaAnalysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA PolymerasesPercze, Krisztina; Meszaros, TamasChemBioChem (2020), 21 (20), 2939-2944CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)One of the pivotal steps in aptamer selection is the amplification of target-specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in-depth study of using 5-indolyl-AA-dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo-) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water-in-oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high-throughput sequence anal. showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries.
- 42Hwang, G. T.; Romesberg, F. E. Unnatural substrate repertoire of A, B, and X family DNA polymerases. J. Am. Chem. Soc. 2008, 130, 14872– 14882, DOI: 10.1021/ja803833hGoogle Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1ehsbzJ&md5=453d01da1b8844dbd1a15674b9612bc7Unnatural Substrate Repertoire of A, B, and X Family DNA PolymerasesHwang, Gil Tae; Romesberg, Floyd E.Journal of the American Chemical Society (2008), 130 (44), 14872-14882CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)As part of an effort to develop unnatural base pairs that are stable and replicable in DNA, we examd. the ability of five different polymerases to replicate DNA contg. four different unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs. The unnatural pairs were developed based on intensive studies using the Klenow fragment of DNA polymerase I from E. coli (Kf) and found to be recognized to varying degrees. The five addnl. polymerases characterized here include family A polymerases from bacteriophage T7 and Thermus aquaticus, family B polymerases from Thermococcus litoralis and Thermococcus 9°N-7, and the family X polymerase, human polymerase β. While we find that some aspects of unnatural base pair recognition are conserved among the polymerases, for example, the pair formed between two d3FB nucleotides is typically well recognized, the detailed recognition of most of the unnatural base pairs is generally polymerase dependent. In contrast, we find that the pair formed between d5SICS and dMMO2 is generally well recognized by all of the polymerases examd., suggesting that the determinants of efficient and general recognition are contained within the geometric and electronic structure of these unnatural nucleobases themselves. The data suggest that while the d3FB:d3FB pair is sufficiently well recognized by several of the polymerases for in vitro applications, the d5SICS:dMMO2 heteropair is likely uniquely promising for in vivo use. T7-mediated replication is esp. noteworthy due to strong mispair discrimination.
- 43Jahiruddin, S.; Datta, A. What sustains the unnatural base pairs (UBPs) with no hydrogen bonds. J. Phys. Chem. B. 2015, 119, 5839– 5845, DOI: 10.1021/acs.jpcb.5b03293Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmvVygtbY%253D&md5=6e8c0394bac5cf300680757f358746a1What Sustains the Unnatural Base Pairs (UBPs) with No Hydrogen BondsJahiruddin, Sk; Datta, AyanJournal of Physical Chemistry B (2015), 119 (18), 5839-5845CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)The recent report of a hydrophobic unnatural base pair (UBP), d5SICS-dNaM, which replicated in DNA PCR and also sustained and synthesized a plasmid inside E. coli genome by Romesberg and coworkers, is intriguing. Quantum chem. calcns. show that the UBPs prefer a slipped parallel configuration to facilitate weak dispersion interactions somewhat similar to the so-called π-stacking interaction. Nevertheless, within a natural DNA tract, classical mol. dynamics simulations show that the backbone and neighboring stacked bases together reorient the UBPs in natural base pair-like planar environment. Our computed structure with an av. end-end distance, dC1'-C1' = 10.7 Å for d5SICS-dNaM is in excellent agreement with available crystal structure (PDB ID: 3SV3, planar UBP with dC1'-C1'crystal = 11.0 Å). Quantum mech. calcns. for the UBP flanked by two natural base-pairs (A-T) on top and on bottom on equilibrated MD structure found large binding energy (ΔE = -74.0 kcal/mol). The present calcns. therefore establish the fact that the hydrophobic UBPs can be stabilized by dispersion interactions with other base pairs in the DNA tract even in the absence of any hydrogen bonding between the UBPs themselves.
- 44Betz, K.; Malyshev, D. A.; Lavergne, T.; Welte, W.; Diederichs, K.; Dwyer, T. J.; Ordoukhanian, P.; Romesberg, F. E.; Marx, A. KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry. Nat. Chem. Biol. 2012, 8, 612– 614, DOI: 10.1038/nchembio.966Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnvVyrurw%253D&md5=3f53105607a419a1d1326125178d682dKlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometryBetz, Karin; Malyshev, Denis A.; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Dwyer, Tammy J.; Ordoukhanian, Phillip; Romesberg, Floyd E.; Marx, AndreasNature Chemical Biology (2012), 8 (7), 612-614CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Many candidate unnatural DNA base pairs have been developed, but some of the best-replicated pairs adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replication for one such pair, dNaM-d5SICS, and show that efficient replication results from the polymerase itself, inducing the required natural-like structure.
- 45Betz, K.; Malyshev, D. A.; Lavergne, T.; Welte, W.; Diederichs, K.; Romesberg, F. E.; Marx, A. Structural insights into DNA replication without hydrogen bonds. J. Am. Chem. Soc. 2013, 135, 18637– 18643, DOI: 10.1021/ja409609jGoogle Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVKjt7zL&md5=59ebd1ad03d32be53c857c76ebf3a2c0Structural insights into DNA replication without hydrogen bondsBetz, Karin; Malyshev, Denis A.; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Romesberg, Floyd E.; Marx, AndreasJournal of the American Chemical Society (2013), 135 (49), 18637-18643CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The genetic alphabet is composed of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chem. potential of DNA. D5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq DNA polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson-Crick-like structure. To understand the remaining steps of replication, the authors now report the characterization of the prechem. complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple postchem. complexes in which the already formed unnatural base pair is positioned at the postinsertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addn. of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the postinsertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are obsd., depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by deintercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.
- 46Hirao, I.; Mitsui, T.; Kimoto, M.; Yokoyama, S. An efficient unnatural base pair for PCR amplification. J. Am. Chem. Soc. 2007, 129, 15549– 15555, DOI: 10.1021/ja073830mGoogle Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlejsbnO&md5=aa94eef33b0951eac30e79775e4623adAn Efficient Unnatural Base Pair for PCR AmplificationHirao, Ichiro; Mitsui, Tsuneo; Kimoto, Michiko; Yokoyama, ShigeyukiJournal of the American Chemical Society (2007), 129 (50), 15549-15555CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Expansion of the genetic alphabet by an unnatural base pair system provides a powerful tool for modern biotechnol. As an alternative to previous unnatural base pairs, we have developed a new pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitropyrrole (Pn), which functions in DNA amplification. Pn more selectively pairs with Ds in replication than another previously reported pairing partner, pyrrole-2-carbaldehyde (Pa). The nitro group of Pn efficiently prevented the mispairing with A. High efficiency and selectivity of the Ds-Pn pair in PCR amplification were achieved by using a substrate mixt. of the γ-amidotriphosphate of Ds and the usual triphosphates of Pn and the natural bases, with Vent DNA polymerase as a 3' to 5' exonuclease-proficient polymerase. After 20 cycles of PCR, the total mutation rate of the Ds-Pn site in an amplified DNA fragment was ∼1%. PCR amplification of DNA fragments contg. the unnatural Ds-Pn pair would be useful for expanded genetic systems in DNA-based biotechnol.
- 47Hashimoto, K.; Fischer, E. C.; Romesberg, F. E. Efforts toward Further Integration of an Unnatural Base Pair into the Biology of a Semisynthetic Organism. J. Am. Chem. Soc. 2021, 143, 8603– 8607, DOI: 10.1021/jacs.1c03860Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXht1ejtbfM&md5=b730f6df8c45a65f5473e661030cf3b4Efforts toward Further Integration of an Unnatural Base Pair into the Biology of a Semisynthetic OrganismHashimoto, Koji; Fischer, Emil C.; Romesberg, Floyd E.Journal of the American Chemical Society (2021), 143 (23), 8603-8607CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have developed semisynthetic organisms (SSOs) that by virtue of a family of synthetic, unnatural base pairs (UBPs), store and retrieve increased information. To date, transcription in the SSOs has relied on heterologous expression of the RNA polymerase from T7 bacteriophage; here, we explore placing transcription under the control of the endogenous host multisubunit RNA polymerase. The results demonstrate that the E. coli RNA polymerase is able to transcribe DNA contg. a UBP and that with the most optimal UBP identified to date it should be possible to select for increased uptake of unnatural triphosphates. These advances should facilitate the creation of next generation SSOs.
- 48Zhang, Y.; Lamb, B. M.; Feldman, A. W.; Zhou, A. X.; Lavergne, T.; Li, L.; Romesberg, F. E. A semisynthetic organism engineered for the stable expansion of the genetic alphabet. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 1317– 1322, DOI: 10.1073/pnas.1616443114Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlCqurs%253D&md5=39e6053994d8c1a47d01833f515c18b0A semisynthetic organism engineered for the stable expansion of the genetic alphabetZhang, Yorke; Lamb, Brian M.; Feldman, Aaron W.; Zhou, Anne Xiaozhou; Lavergne, Thomas; Li, Lingjun; Romesberg, Floyd E.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (6), 1317-1322CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)All natural organisms store genetic information in a four-letter, two-base-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escherichia coli grown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA contg. a single dNaM-d5SICS UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used a more chem. optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet.
- 49Feldman, A. W.; Romesberg, F. E. In vivo structure-activity relationships and optimization of an unnatural base pair for replication in a semi-synthetic organism. J. Am. Chem. Soc. 2017, 139, 11427– 11433, DOI: 10.1021/jacs.7b03540Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlShs7fE&md5=f86f88442184302b1777b217ea25c0abIn Vivo Structure-Activity Relationships and Optimization of an Unnatural Base Pair for Replication in a Semi-Synthetic OrganismFeldman, Aaron W.; Romesberg, Floyd E.Journal of the American Chemical Society (2017), 139 (33), 11427-11433CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)In an effort to expand the genetic alphabet and create semi-synthetic organisms (SSOs) that store and retrieve increased information, we have developed the unnatural base pairs (UBPs) dNaM and d5SICS or dTPT3 (dNaM-d5SICS and dNaM-dTPT3). The UBPs form based on hydrophobic and packing forces, as opposed to complementary hydrogen bonding, and while they are both retained within the in vivo environment of an Escherichia coli SSO, their development was based on structure-activity relationship (SAR) data generated in vitro. To address the likely possibility of different requirements of the in vivo environment, we screened 135 candidate UBPs for optimal performance in the SSO. Interestingly, we find that in vivo SARs differ from those collected in vitro, and most importantly, we identify four UBPs whose retention in the DNA of the SSO is higher than that of dNaM-dTPT3, which was previously the most promising UBP identified. The identification of these four UBPs further demonstrates that when optimized, hydrophobic and packing forces may be used to replace the complementary hydrogen bonding used by natural pairs and represents a significant advance in our continuing efforts to develop SSOs that store and retrieve more information than natural organisms.
- 50Seo, Y. J.; Hwang, G. T.; Ordoukhanian, P.; Romesberg, F. E. Optimization of an unnatural base pair toward natural-like replication. J. Am. Chem. Soc. 2009, 131, 3246– 3252, DOI: 10.1021/ja807853mGoogle Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXit1eksbc%253D&md5=0896876c67103ffd1845f2313624bd8bOptimization of an Unnatural Base Pair toward Natural-Like ReplicationSeo, Young Jun; Hwang, Gil Tae; Ordoukhanian, Phillip; Romesberg, Floyd E.Journal of the American Chemical Society (2009), 131 (9), 3246-3252CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Predominantly hydrophobic unnatural nucleotides that selectively pair within duplex DNA as well as during polymerase-mediated replication have recently received much attention as the cornerstone of efforts to expand the genetic alphabet. We recently reported the results of a screen and subsequent lead hit optimization that led to identification of the unnatural base pair formed between the nucleotides dMMO2 and d5SICS. This unnatural base pair is replicated by the Klenow fragment of Escherichia coli DNA polymerase I with better efficiency and fidelity than other candidates reported in the literature. However, its replication remains significantly less efficient than a natural base pair, and further optimization is necessary for its practical use. To better understand and optimize the slowest step of replication of the unnatural base pair, the insertion of dMMO2 opposite d5SICS, we synthesized two dMMO2 derivs., d5FM and dNaM, which differ from the parent nucleobase in terms of shape, hydrophobicity, and polarizability. We find that both derivs. are inserted opposite d5SICS more efficiently than dMMO2 and that overall the corresponding unnatural base pairs are generally replicated with higher efficiency and fidelity than the pair between dMMO2 and d5SICS. In fact, in the case of the dNaM:d5SICS heteropair, the efficiency of each individual step of replication approaches that of a natural base pair, and the min. overall fidelity ranges from 103 to 104. In addn., the data allow us to propose a generalized model of unnatural base pair replication, which should aid in further optimization of the unnatural base pair and possibly in the design of addnl. unnatural base pairs that are replicated with truly natural-like efficiency and fidelity.
- 51Malyshev, D. A.; Dhami, K.; Quach, H. T.; Lavergne, T.; Ordoukhanian, P.; Torkamani, A.; Romesberg, F. E. Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 12005– 12010, DOI: 10.1073/pnas.1205176109Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1GrtL3M&md5=a3e02f5c8ccda2071120e01ce1b059d1Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabetMalyshev, Denis A.; Dhami, Kirandeep; Quach, Henry T.; Lavergne, Thomas; Ordoukhanian, Phillip; Torkamani, Ali; Romesberg, Floyd E.Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (30), 12005-12010, S12005/1-S12005/31CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chem. and synthetic biol. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA contg. one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under std. conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates contg. the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that for PCR and PCR-based applications, d5SICS-dNaM is functionally equiv. to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.
- 52Sharma, P.; Manderville, R. A.; Wetmore, S. D. Structural and energetic characterization of the major DNA adduct formed from the food mutagen ochratoxin A in the NarI hotspot sequence: influence of adduct ionization on the conformational preferences and implications for the NER propensity. Mutagenic. The Significance of Internal Communication in the Management of Successful Change. Nucleic Acids Res. 2014, 42, 11831– 11845, DOI: 10.1093/nar/gku821Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVChtr%252FF&md5=b92a626995c4d1f98cfaa6b77a58c787Structural and energetic characterization of the major DNA adduct formed from the food mutagen ochratoxin A in the NarI hotspot sequence: influence of adduct ionization on the conformational preferences and implications for the NER propensitySharma, Purshotam; Manderville, Richard A.; Wetmore, Stacey D.Nucleic Acids Research (2014), 42 (18), 11831-11845CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The nephrotoxic food mutagen ochratoxin A (OTA) produces DNA adducts in rat kidneys, the major lesion being the C8-linked-2'-deoxyguanosine adduct (OTB-dG). Although research on other adducts stresses the importance of understanding the structure of the assocd. adducted DNA, site-specific incorporation of OTB-dG into DNA has yet to be attempted. The present work uses a robust computational approach to det. the conformational preferences of OTB-dG in three ionization states at three guanine positions in the NarI recognition sequence opposite cytosine. Representative adducted DNA helixes were derived from over 2160 ns of simulation and ranked via free energies. For the first time, a close energetic sepn. between three distinct conformations is highlighted, which indicates OTA-adducted DNA likely adopts a mixt. of conformations regardless of the sequence context. Nevertheless, the preferred conformation depends on the flanking bases and ionization state due to deviations in discrete local interactions at the lesion site. The structural characteristics of the lesion thus discerned have profound implications regarding its repair propensity and mutagenic outcomes, and support recent expts. suggesting the induction of double-strand breaks and deletion mutations upon OTA exposure. This combined structural and energetic characterization of the OTB-dG lesion in DNA will encourage future biochem. expts. on this potentially genotoxic lesion.
- 53Flamme, M.; Röthlisberger, P.; Levi-Acobas, F.; Chawla, M.; Oliva, R.; Cavallo, L.; Gasser, G.; Marlière, P.; Herdewijn, P.; Hollenstein, M. Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside Triphosphate. ACS Chem. Biol. 2020, 15, 2872– 2884, DOI: 10.1021/acschembio.0c00396Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFagtLjK&md5=98c9d3978e55cb62ddb9db007684bf29Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside TriphosphateFlamme, Marie; Rothlisberger, Pascal; Levi-Acobas, Fabienne; Chawla, Mohit; Oliva, Romina; Cavallo, Luigi; Gasser, Gilles; Marliere, Philippe; Herdewijn, Piet; Hollenstein, MarcelACS Chemical Biology (2020), 15 (11), 2872-2884CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)The expansion of the genetic alphabet with addnl., unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biol. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymic construction of a silver-mediated base pair (dImC-AgI-dPurP) as well as a two-step protocol for the synthesis of DNA duplexes contg. such an artificial metal base pair. Guided by DFT calcns., we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymic synthesis of the dImC-AgI-dPurP artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.
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This article is cited by 3 publications.
- Floyd E. Romesberg. Discovery, implications and initial use of semi-synthetic organisms with an expanded genetic alphabet/code. Philosophical Transactions of the Royal Society B: Biological Sciences 2023, 378
(1871)
https://doi.org/10.1098/rstb.2022.0030
- Marcel Hollenstein. Enzymatic Synthesis of Base-Modified Nucleic Acids. 2023, 1-39. https://doi.org/10.1007/978-981-16-1313-5_23-1
- William Whitaker, Katya E. Moncrieff, Cate S. Anstöter, Natalie G. K. Wong, Jacob A. Berenbeim, Caroline E. H. Dessent. Probing the electronic relaxation pathways and photostability of the synthetic nucleobase Z
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Abstract
Figure 1
Figure 1. (A) Design and structures of TPT3-type unnatural base nucleosides. (B) UV–vis spectra of the synthesized TPT3-type unnatural base nucleosides.
Figure 2
Figure 2. Photostability of dTPT3 and its analogues. (A) Photostability of dTPT3; (B) photostability of dTPT4; (C) photostability of dTAT1; (D) photostability of d4TFP.
Figure 3
Figure 3. Kinetics analysis and sequencing analysis of the PCR products. (A) Kf-mediated primer extension and sequences used in this study; (B) representative gel for the incorporation assay; (C) representative gel for the extension assay. (D) Sequencing analysis of the PCR products after 36 cycles by OneTaq polymerase.
Figure 4
Figure 4. Optimized base pairs between dNaM and dTPT3 as well as its analogues (left). Optimal distances in the gas phase of the key atoms and C1′–C1′ distances. Oxygen, sulfur, and nitrogen are colored red, yellow, and blue, respectively. The electrostatic potential (ESP) maps (right) describe the electron density distribution; the red area represents the electron-rich area; and the green area represents the electron-deficient area.
Figure 5
Figure 5. Intracellular UBP replication and physiology of the E. coli cells after incubation for 17 h. (A) Overview of plasmid construction and transforming. X: dNaM; Y: dTPT3; or dTAT1. Color indicates overlapped sequences. The plasmids containing UBPs or PtNTT2 were transformed into E. coli BL 21 (DE3) together. (B) Retention of the UBPs. (C) Growth of E. coli cells cultured with UBPs. (D) Growth of E. coli cells cultured with dTPT3 or dTAT1. (E) ROS content of E. coli cells cultured with dTPT3 or dTAT1. Data represents the mean ± S.D. (n = 3), Student’s t test, **p < 0.01.
References
This article references 53 other publications.
- 1Piccirilli, J. A.; Benner, S. A.; Krauch, T.; Moroney, S. E. Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature 1990, 343, 33– 37, DOI: 10.1038/343033a01https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXitFamu7k%253D&md5=1abf50139303a7ffa31262cdeb736fe5Enzymic incorporation of a new base pair into DNA and RNA extends the genetic alphabetPiccirilli, Joseph A.; Krauch, Tilman; Moroney, Simon E.; Benner, Steven A.Nature (London, United Kingdom) (1990), 343 (6253), 33-7CODEN: NATUAS; ISSN:0028-0836.A new Watson-Crick base pair [κ-xanthine or 7-Me oxyformycin B, where κ = 3-β-D-ribofuranosyl-(2,6-diaminopyrimidine) with a H bonding pattern different from that in the A·T and G·C base pairs, is incorporated into duplex DNA and RNA by DNA and RNA polymerases and expands the genetic alphabet from 4 to 6 letters. This expansion could lead to RNAs with greater diversity in functional groups and greater catalytic potential.
- 2Malyshev, D. A.; Dhami, K.; Lavergne, T.; Chen, T.; Dai, N.; Foster, J. M.; Corrêa, I. R.; Romesberg, F. E. A semi-synthetic organism with an expanded genetic alphabet. Nature 2014, 509, 385– 388, DOI: 10.1038/nature133142https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXotVyqtb8%253D&md5=97b4b184cda52cc809b1705e5e88ad8eA semi-synthetic organism with an expanded genetic alphabetMalyshev, Denis A.; Dhami, Kirandeep; Lavergne, Thomas; Chen, Tingjian; Dai, Nan; Foster, Jeremy M.; Correa, Ivan R.; Romesberg, Floyd E.Nature (London, United Kingdom) (2014), 509 (7500), 385-388CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA contg. the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid contg. d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
- 3Kimoto, M.; Hirao, I. Genetic alphabet expansion technology by creating unnatural base pairs. Chem. Soc. Rev. 2020, 49, 7602– 7626, DOI: 10.1039/D0CS00457J3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFyit7bL&md5=8bb0ea148d02d47f184b412bd0e616feGenetic alphabet expansion technology by creating unnatural base pairsKimoto, Michiko; Hirao, IchiroChemical Society Reviews (2020), 49 (21), 7602-7626CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Recent advancements in the creation of artificial extra base pairs (unnatural base pairs, UBPs) are opening the door to a new research area, xenobiol., and genetic alphabet expansion technologies. UBPs that function as third base pairs in replication, transcription, and/or translation enable the site-specific incorporation of novel components into DNA, RNA, and proteins. Here, we describe the UBPs developed by three research teams and their application in PCR-based diagnostics, high-affinity DNA aptamer generation, site-specific labeling of RNAs, semi-synthetic organism creation, and unnatural-amino-acid-contg. protein synthesis.
- 4Ledbetter, M. P.; Malyshev, D. A.; Romesberg, F. E. Site-Specific Labeling of DNA via PCR with an Expanded Genetic Alphabet. Methods Mol. Biol. 2019, 1973, 193– 212, DOI: 10.1007/978-1-4939-9216-4_134https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVWrurk%253D&md5=a793c10929b681f4e69b91210933647aSite-specific labeling of DNA via PCR with an expanded genetic alphabetLedbetter, Michael P.; Malyshev, Denis A.; Romesberg, Floyd E.Methods in Molecular Biology (New York, NY, United States) (2019), 1973 (Non-Natural Nucleic Acids), 193-212CODEN: MMBIED; ISSN:1940-6029. (Springer)A review. The polymerase chain reaction (PCR) is a universal and essential tool in mol. biol. and biotechnol., but it is generally limited to the amplification of DNA with the four-letter genetic alphabet. Here, we describe PCR amplification with a six-letter alphabet that includes the two natural dA-dT and dG-dC base pairs and an unnatural base pair (UBP) formed between the synthetic nucleotides dNaM and d5SICS or dTPT3 or analogs of these synthetic nucleotides modified with linkers that allow for the site-specific labeling of the amplified DNA with different functional groups. Under std. conditions, the six-letter DNA may be amplified with high efficiency and with greater than 99.9% fidelity. This allows for the efficient prodn. of DNA site-specifically modified with different functionalities of interest for use in a wide range of applications.
- 5Kimoto, M.; Mitsui, T.; Harada, Y.; Sato, A.; Yokoyama, S.; Hirao, I. Fluorescent probing for RNA molecules by an unnatural base-pair system. Nucleic Acids Res. 2007, 35, 5360– 5369, DOI: 10.1093/nar/gkm5085https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1ygtb7N&md5=9229005556346b52fa5f42fba40a7200Fluorescent probing for RNA molecules by an unnatural base-pair systemKimoto, Michiko; Mitsui, Tsuneo; Harada, Yoko; Sato, Akira; Yokoyama, Shigeyuki; Hirao, IchiroNucleic Acids Research (2007), 35 (16), 5360-5369CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl) purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5'-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates. The fluorescent intensity of s in RNA mols. changes according to the structural environment. The site-specific s labeling of RNA hairpins and tRNA mols. provided characteristic fluorescent profiles, depending on the labeling sites, temp. and Mg2+ concn. The Pa-contg. DNA templates can be amplified by PCR using 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), another pairing partner of Pa. This site-specific fluorescent probing by the unnatural pair system including the s-Pa and Ds-Pa pairs provides a powerful tool for studying the dynamics of the local structural features of 3D RNA mols. and their intra- and intermol. interactions.
- 6Kimoto, M.; Matsunaga, K. I.; Hirao, I. Evolving Aptamers with Unnatural Base Pairs. Curr. Protoc. Chem. Biol. 2017, 9, 315– 339, DOI: 10.1002/cpch.316https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjtVOmsrw%253D&md5=5d47ec6b5ec780052ef39872705a9f42Evolving Aptamers with Unnatural Base PairsKimoto, Michiko; Matsunaga, Ken-ichiro; Hirao, IchiroCurrent Protocols in Chemical Biology (2017), 9 (4), 315-339CODEN: CPCBD9; ISSN:2160-4762. (John Wiley & Sons, Inc.)A novel technol., genetic alphabet expansion, has rapidly advanced through the successful creation of unnatural base pairs that function as a third base pair in replication. Recently, genetic alphabet expansion has been applied to some practical areas. Among them, the application to DNA aptamer generation is a good example of the broad utility of this technol. A hydrophobic unnatural base pair, Ds-Px, which exhibits high fidelity in replication as a third base pair, has been applied to an evolutionary engineering method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) to generate DNA aptamers that bind to targets. A few Ds bases in DNA aptamers significantly increase the binding affinity to targets, enabling the use of DNA aptamers as an alternative to antibodies. This protocol describes the ExSELEX (genetic alphabet Expansion for SELEX) method to generate Ds-contg. DNA aptamers. © 2017 by John Wiley & Sons, Inc.
- 7Hoshika, S.; Leal, N. A.; Kim, M.-J.; Kim, M.-S.; Karalkar, N. B.; Kim, H.-J.; Bates, A. M.; Watkins, N. E., Jr.; SantaLucia, H. A.; Meyer, A. J.; DasGupta, S.; Piccirilli, J. A.; Ellington, A. D.; SantaLucia, J., Jr.; Georgiadis, M. M.; Benner, S. A. Hachimoji DNA and RNA: a genetic system with eight building blocks. Science 2019, 363, 884– 887, DOI: 10.1126/science.aat09717https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjt1elu7c%253D&md5=1ae87a691cfdfbe36a819d4782c305b9Hachimoji DNA and RNA: A genetic system with eight building blocksHoshika, Shuichi; Leal, Nicole A.; Kim, Myong-Jung; Kim, Myong-Sang; Karalkar, Nilesh B.; Kim, Hyo-Joong; Bates, Alison M.; Watkins, Norman E., Jr.; SantaLucia, Holly A.; Meyer, Adam J.; DasGupta, Saurja; Piccirilli, Joseph A.; Ellington, Andrew D.; SantaLucia, John, Jr.; Georgiadis, Millie M.; Benner, Steven A.Science (Washington, DC, United States) (2019), 363 (6429), 884-887CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodn. stability, and stereoregular building blocks that fit a Schroedinger aperiodic crystal. Measured thermodn. parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information d. of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of mol. structures that might support life, including life throughout the cosmos.
- 8Kimoto, M.; Yamashige, R.; Matsunaga, K.; Yokoyama, S.; Hirao, I. Generation of high-affinity DNA aptamers using an expanded genetic alphabet. Nat. Biotechnol. 2013, 31, 453– 457, DOI: 10.1038/nbt.25568https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltlOhur4%253D&md5=068493fd06e1537c468ed340055ce3ecGeneration of high-affinity DNA aptamers using an expanded genetic alphabetKimoto, Michiko; Yamashige, Rie; Matsunaga, Ken-ichiro; Yokoyama, Shigeyuki; Hirao, IchiroNature Biotechnology (2013), 31 (5), 453-457CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)DNA aptamers produced with natural or modified natural nucleotides often lack the desired binding affinity and specificity to target proteins. Here we describe a method for selecting DNA aptamers contg. the four natural nucleotides and an unnatural nucleotide with the hydrophobic base 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). We incorporated up to three Ds nucleotides in a random sequence library, which is expected to increase the chem. and structural diversity of the DNA mols. Selection expts. against two human target proteins, vascular endothelial cell growth factor-165 (VEGF-165) and interferon-γ (IFN-γ), yielded DNA aptamers that bind with KD values of 0.65 pM and 0.038 nM, resp., affinities that are >100-fold improved over those of aptamers contg. only natural bases. These results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids.
- 9Manandhar, M.; Chun, E.; Romesberg, F. E. Genetic code expansion: inception, development, commercialization. J. Am. Chem. Soc. 2021, 143, 4859– 4878, DOI: 10.1021/jacs.0c119389https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXmvFarsb0%253D&md5=013699b4fc4a239c1424b6d7786e6eb5Genetic Code Expansion: Inception, Development, CommercializationManandhar, Miglena; Chun, Eugene; Romesberg, Floyd E.Journal of the American Chemical Society (2021), 143 (13), 4859-4878CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A review. Virtually all natural proteins are built from only 20 amino acids, and while this makes possible all the functions they perform, the ability to encode other amino acids selected for specific purposes promises to enable the discovery and prodn. of proteins with novel functions, including therapeutic proteins with more optimal drug-like properties. The field of genetic code expansion (GCE) has for years enabled the prodn. of such proteins for academic purposes and is now transitioning to commercialization for the prodn. of more optimal protein therapeutics. Focusing on E. coli, we review the history and current state of the field. We also provide a review of the first generation commercialization efforts, the lessons learned, and how those lessons are guiding new efforts. With continued academic and industrial progress, GCE methodologies promise to make possible the routine optimization of proteins for therapeutic use in a way that has only previously been possible with small-mol. therapeutics.
- 10Feldman, A. W.; Dien, V. T.; Karadeema, R. J.; Fischer, E. C.; You, Y.; Anderson, B. A.; Krishnamurthy, R.; Chen, J. S.; Li, L.; Romesberg, F. E. Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism. J. Am. Chem. Soc. 2019, 141, 10644– 10653, DOI: 10.1021/jacs.9b0207510https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1eru7%252FL&md5=546bdf9cea64eed550829034588078caOptimization of Replication, Transcription, and Translation in a Semi-Synthetic OrganismFeldman, Aaron W.; Dien, Vivian T.; Karadeema, Rebekah J.; Fischer, Emil C.; You, Yanbo; Anderson, Brooke A.; Krishnamurthy, Ramanarayanan; Chen, Jason S.; Li, Lingjun; Romesberg, Floyd E.Journal of the American Chemical Society (2019), 141 (27), 10644-10653CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins contg. noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogs. Importantly, this includes the first in vivo structure-activity relationship (SAR) anal. of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were obsd., and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homol. to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins contg. multiple, proximal ncAAs.
- 11Zhou, A. X.-Z.; Sheng, K.; Feldman, A. W.; Romesberg, F. E. Progress toward Eukaryotic Semisynthetic Organisms: Translation of Unnatural Codons. J. Am. Chem. Soc. 2019, 141, 20166– 20170, DOI: 10.1021/jacs.9b0908011https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlymtrzI&md5=f27e1a7d3a75e53648b2fdc724f46a47Progress toward Eukaryotic Semisynthetic Organisms: Translation of Unnatural CodonsZhou, Anne Xiao-Zhou; Sheng, Kai; Feldman, Aaron W.; Romesberg, Floyd E.Journal of the American Chemical Society (2019), 141 (51), 20166-20170CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have created a bacterial semisynthetic organism (SSO) that retains an unnatural base pair (UBP) in its DNA, transcribes it into mRNA and tRNA with cognate unnatural codons and anticodons, and after the tRNA is charged with a noncanonical amino acid, synthesizes proteins contg. the noncanonical amino acid. Here, we report the first progress toward the creation of eukaryotic SSOs. After demonstrating proof-of-concept with human HEK293 cells, we show that a variety of different unnatural codon-anticodon pairs can efficiently mediate the synthesis of unnatural proteins in CHO cells. Interestingly, we find that there are both similarities and significant differences between how the prokaryotic and eukaryotic ribosomes recognize the UBP, with the eukaryotic ribosome appearing more tolerant. The results represent the first progress toward eukaryotic SSOs and in fact, suggest that such SSOs might be able to retain more unnatural information than their bacterial counterparts.
- 12Fischer, E. C.; Hashimoto, K.; Zhang, Y.; Feldman, A. W.; Dien, V. T.; Karadeema, R. J.; Adhikary, R.; Ledbetter, M. P.; Krishnamurthy, R.; Romesberg, F. E. New codons for efficient production of unnatural proteins in a semisynthetic organism. Nat. Chem. Biol. 2020, 16, 570– 576, DOI: 10.1038/s41589-020-0507-z12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsFShsL4%253D&md5=567a5f0d421422c14ff754fa23e30f84New codons for efficient production of unnatural proteins in a semisynthetic organismFischer, Emil C.; Hashimoto, Koji; Zhang, Yorke; Feldman, Aaron W.; Dien, Vivian T.; Karadeema, Rebekah J.; Adhikary, Ramkrishna; Ledbetter, Michael P.; Krishnamurthy, Ramanarayanan; Romesberg, Floyd E.Nature Chemical Biology (2020), 16 (5), 570-576CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Abstr.: Natural organisms use a four-letter genetic alphabet that makes available 64 triplet codons, of which 61 are sense codons used to encode proteins with the 20 canonical amino acids. We have shown that the unnatural nucleotides dNaM and dTPT3 can pair to form an unnatural base pair (UBP) and allow for the creation of semisynthetic organisms (SSOs) with addnl. sense codons. Here, we report a systematic anal. of the unnatural codons. We identify nine unnatural codons that can produce unnatural protein with nearly complete incorporation of an encoded noncanonical amino acid (ncAA). We also show that at least three of the codons are orthogonal and can be simultaneously decoded in the SSO, affording the first 67-codon organism. The ability to incorporate multiple, different ncAAs site specifically into a protein should now allow the development of proteins with novel activities, and possibly even SSOs with new forms and functions. [graphic not available: see full text]. The GenBank accesion nos. were added from NCBI database MN882182-MN882190.
- 13Hamashima, K.; Kimoto, M.; Hirao, I. Creation of unnatural base pairs for genetic alphabet expansion toward synthetic xenobiology. Curr. Opin. Chem. Biol. 2018, 46, 108– 114, DOI: 10.1016/j.cbpa.2018.07.01713https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhsVSmtLfJ&md5=9d539f07e70cccfa4372d13fc3c026f4Creation of unnatural base pairs for genetic alphabet expansion toward synthetic xenobiologyHamashima, Kiyofumi; Kimoto, Michiko; Hirao, IchiroCurrent Opinion in Chemical Biology (2018), 46 (), 108-114CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Artificial extra base pairs (unnatural base pairs, UBPs) expand the genetic alphabet of DNA, thus broadening entire biol. systems in the central dogma. UBPs function as third base pairs in replication, transcription, and/or translation, and have created a new research area, synthetic xenobiol., providing genetic engineering tools to generate novel DNAs, RNAs, and proteins with increased functionalities. Several UBPs have been developed and applied to PCR technol., DNA aptamer generation, and semi-synthetic organism creation. Among them, we developed a series of UBPs and demonstrated unique quant. PCR and high-affinity DNA aptamer generation methods.
- 14Saito-Tarashima, N.; Minakawa, N. Unnatural Base Pairs for Synthetic Biology. Chem. Pharm. Bull. 2018, 66, 132– 138, DOI: 10.1248/cpb.c17-0068514https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVKrtrbF&md5=0bb731d9dec57019c159f4b02b35bdacUnnatural base pairs for synthetic biologySaito-Tarashima, Noriko; Minakawa, NoriakiChemical & Pharmaceutical Bulletin (2018), 66 (2), 132-138CODEN: CPBTAL; ISSN:0009-2363. (Pharmaceutical Society of Japan)A review. In this review, we have summarized the research effort into the development of unnatural base pairs beyond std. Watson-Crick (WC) base pairs for synthetic biol. Prior to introducing our research results, we present investigations by four outstanding groups in the field. Their research results demonstrate the importance of shape complementarity and stacking ability as well as hydrogen-bonding (H-bonding) pat- terns for unnatural base pairs. On the basis of this research background, we developed unnatural base pairs consisting of imidazo[5'4':4.5]pyrido[2,3-d]pyrimidines and 1,8-naphthyridines, i.e., I in : Na pairs. Since Im bases are recognized as ring-expanded purines and Na bases are recognized as ring-expanded pyrimidines, I m :Na pairs are expected to satisfy the criteria of shape complementarity and enhanced stacking ability. In addn., these pairs have four non-canonical H-bonds. Because of these preferable properties, ImNN:NaOO, one of the Im:Na pairs, is recognized as a complementary base pair in not only single nucleotide insertion, but also the PCR.
- 15Marx, A.; Betz, K. The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases. Chem. – Eur. J. 2020, 26, 3446– 3463, DOI: 10.1002/chem.20190352515https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXht1Gqsrs%253D&md5=7ef06701ea4cb58153a780f96ef717c4The Structural Basis for Processing of Unnatural Base Pairs by DNA PolymerasesMarx, Andreas; Betz, KarinChemistry - A European Journal (2020), 26 (16), 3446-3463CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Unnatural base pairs (UBPs) greatly increase the diversity of DNA and RNA, furthering their broad range of mol. biol. and biotechnol. approaches. Different candidates have been developed whereby alternative hydrogen-bonding patterns and hydrophobic and packing interactions have turned out to be the most promising base-pairing concepts to date. The key in many applications is the highly efficient and selective acceptance of artificial base pairs by DNA polymerases, which enables amplification of the modified DNA. In this Review, computational as well as exptl. studies that were performed to characterize the pairing behavior of UBPs in free duplex DNA or bound to the active site of KlenTaq DNA polymerase are highlighted. The structural studies, on the one hand, elucidate how base pairs lacking hydrogen bonds are accepted by these enzymes and, on the other hand, highlight the influence of one or several consecutive UBPs on the structure of a DNA double helix. Understanding these concepts facilitates optimization of future UBPs for the manifold fields of applications.
- 16Li, L.; Degardin, M.; Lavergne, T.; Malyshev, D. A.; Dhami, K.; Ordoukhanian, P.; Romesberg, F. E. Natural-like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applications. J. Am. Chem. Soc. 2014, 136, 826– 829, DOI: 10.1021/ja408814g16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1KktLnO&md5=5d290f34aab307b9a965dfca3386dcf7Natural-like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applicationsLi, Lingjun; Degardin, Melissa; Lavergne, Thomas; Malyshev, Denis A.; Dhami, Kirandeep; Ordoukhanian, Phillip; Romesberg, Floyd E.Journal of the American Chemical Society (2014), 136 (3), 826-829CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The authors synthesized a panel of unnatural base pairs whose pairing depends on hydrophobic and packing forces and identify dTPT3 (I)-dNaM, which is PCR amplified with a natural base pair-like efficiency and fidelity. In addn., the I scaffold is uniquely tolerant of attaching a propargyl amine linker, resulting in the dTPT3PA-dNaM pair, which is amplified only slightly less well. The identification of I represents significant progress toward developing an unnatural base pair for the in vivo expansion of an organism's genetic alphabet and for a variety of in vitro biotechnol. applications where it is used to site-specifically label amplified DNA, and it also demonstrates for the first time that hydrophobic and packing forces are sufficient to mediate natural-like replication.
- 17Zhang, Y.; Ptacin, J. L.; Fischer, E. C.; Aerni, H. R.; Caffaro, C. E.; San Jose, K.; Feldman, A. W.; Turner, C. R.; Romesberg, F. E. A semi-synthetic organism that stores and retrieves increased genetic information. Nature 2017, 551, 644– 647, DOI: 10.1038/nature2465917https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWhtL7L&md5=d04861904783b1a852d4089b28f09acfA semi-synthetic organism that stores and retrieves increased genetic informationZhang, Yorke; Ptacin, Jerod L.; Fischer, Emil C.; Aerni, Hans R.; Caffaro, Carolina E.; San Jose, Kristine; Feldman, Aaron W.; Turner, Court R.; Romesberg, Floyd E.Nature (London, United Kingdom) (2017), 551 (7682), 644-647CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Since at least the last common ancestor of all life on Earth, genetic information has been stored in a four-letter alphabet that is propagated and retrieved by the formation of two base pairs. The central goal of synthetic biol. is to create new life forms and functions, and the most general route to this goal is the creation of semi-synthetic organisms whose DNA harbors two addnl. letters that form a third, unnatural base pair. Previous efforts to generate such semi-synthetic organisms culminated in the creation of a strain of Escherichia coli that, by virtue of a nucleoside triphosphate transporter from Phaeodactylum tricornutum, imports the requisite unnatural triphosphates from its medium and then uses them to replicate a plasmid contg. the unnatural base pair dNaM-dTPT3. Although the semi-synthetic organism stores increased information when compared to natural organisms, retrieval of the information requires in vivo transcription of the unnatural base pair into mRNA and tRNA, aminoacylation of the tRNA with a non-canonical amino acid, and efficient participation of the unnatural base pair in decoding at the ribosome. Here we report the in vivo transcription of DNA contg. dNaM and dTPT3 into mRNAs with two different unnatural codons and tRNAs with cognate unnatural anticodons, and their efficient decoding at the ribosome to direct the site-specific incorporation of natural or non-canonical amino acids into superfolder green fluorescent protein. The results demonstrate that interactions other than hydrogen bonding can contribute to every step of information storage and retrieval. The resulting semi-synthetic organism both encodes and retrieves increased information and should serve as a platform for the creation of new life forms and functions.
- 18Kimoto, M.; Kawai, R.; Mitsui, T.; Yokoyama, S.; Hirao, I. An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules. Nucleic Acids Res. 2009, 37, e14 DOI: 10.1093/nar/gkn95618https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlGksL8%253D&md5=8812c6b48c1a2c611efdd4f77afda742An unnatural base pair system for efficient PCR amplification and functionalization of DNA moleculesKimoto, Michiko; Kawai, Rie; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, IchiroNucleic Acids Research (2009), 37 (2), e14/1-e14/9CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates contg. unnatural bases, natural and unnatural base triphosphates, and a 3'→5' exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library contg. the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) contg. the unnatural base pair can be amplified 107-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA mols. from mixts. with excess amts. (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.
- 19Yang, Z.; Sismour, A. M.; Sheng, P.; Puskar, N. L.; Benner, S. A. Enzymatic incorporation of a third nucleobase pair. Nucleic Acids Res. 2007, 35, 4238– 4249, DOI: 10.1093/nar/gkm39519https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXos12nsr4%253D&md5=9b802c5f84a6e199ced685bb6fa31d72Enzymatic incorporation of a third nucleobase pairYang, Zunyi; Sismour, A. Michael; Sheng, Pinpin; Puskar, Nyssa L.; Benner, Steven A.Nucleic Acids Research (2007), 35 (13), 4238-4249CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)DNA polymerases are identified that copy a non-std. nucleotide pair joined by a hydrogen bonding pattern different from the patterns joining the dA:T and dG:dC pairs. 6-Amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) implements the non-std. 'small' donor-donor-acceptor (pyDDA) hydrogen bonding pattern. 2-Amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP) implements the 'large' acceptor-acceptor-donor (puAAD) pattern. These nucleobases were designed to present electron d. to the minor groove, d. hypothesized to help det. specificity for polymerases. Consistent with this hypothesis, both dZTP and dPTP are accepted by many polymerases from both Families A and B. Further, the dZ:dP pair participates in PCR reactions catalyzed by Taq, Vent (exo-) and Deep Vent (exo-) polymerases, with 94.4%, 97.5% and 97.5%, resp., retention per round. The dZ:dP pair appears to be lost principally via transition to a dC:dG pair. This is consistent with a mechanistic hypothesis that deprotonated dZ (presenting a pyDAA pattern) complements dG (presenting a puADD pattern), while protonated dC (presenting a pyDDA pattern) complements dP (presenting a puAAD pattern). This hypothesis, grounded in the Watson-Crick model for nucleobase pairing, was confirmed by studies of the pH-dependence of mismatching. The dZ:dP pair and these polymerases, should be useful in dynamic architectures for sequencing, mol.-, systems- and synthetic-biol.
- 20Yang, Z.; Chen, F.; Alvarado, J. B.; Benner, S. A. Amplification, mutation, and sequencing of a six-letter synthetic genetic system. J. Am. Chem. Soc. 2011, 133, 15105– 15112, DOI: 10.1021/ja204910n20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFaks7zL&md5=cc4561e9527ca359c82494deb9e0be2dAmplification, mutation, and sequencing of a six-letter synthetic genetic systemYang, Zun-Yi; Chen, Fei; Alvarado, J. Brian; Benner, Steven A.Journal of the American Chemical Society (2011), 133 (38), 15105-15112CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The next goals in the development of a synthetic biol. that uses artificial genetic systems will require chem.-biol. combinations that allow the amplification of DNA contg. any no. of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technol. is needed to sequence artificial genetic DNA mols. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four std. nucleotides (G, A, C, and T) and two addnl. nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theor. cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.
- 21Ashwood, B.; Pollum, M.; Crespo-Hernández, C. E. Can a Six-Letter Alphabet Increase the Likelihood of Photochemical Assault to the Genetic Code?. Chem. – Eur. J. 2016, 22, 16648– 16656, DOI: 10.1002/chem.20160216021https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1ClsLvM&md5=cc289085cffc83044b2879a95b5f4456Can a Six-Letter Alphabet Increase the Likelihood of Photochemical Assault to the Genetic Code?Ashwood, Brennan; Pollum, Marvin; Crespo-Hernandez, Carlos E.Chemistry - A European Journal (2016), 22 (46), 16648-16656CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)In 2014, two unnatural nucleosides, d5SICS and dNaM, were shown to selectively base pair and replicate with high fidelity in a modified strain of E. coli, thus effectively expanding its genetic alphabet from four to six letters. More recently, a significant redn. in cell proliferation was reported in cells cultured with d5SICS, and putatively with dNaM, upon exposure to brief periods of near-visible radiation. The photosensitizing properties of the lowest-energy excited triplet state of both d5SICS and dNaM were implicated in their cytotoxicity. Importantly, however, the excited-state mechanisms by which near-visible excitation populates the triplet states of d5SICS and dNaM are currently unknown. In this study, steady-state and time-resolved spectroscopies are combined with quantum-chem. calcns. in order to reveal the excited-state relaxation mechanisms leading to efficient population of the triplet states in these unnatural nucleosides in soln. It is shown that excitation of d5SICS or dNaM with near-visible light leads overwhelmingly to ultrafast population of their triplet states on the femtosecond time scale. The results presented in this work lend strong support to the proposal that photoexcitation of these unnatural nucleosides can accelerate oxidatively generated damage to DNA and other biomols. within the cellular environment.
- 22Bhattacharyya, K.; Datta, A. Visible-Light-Mediated Excited State Relaxation in Semi-Synthetic Genetic Alphabet: d5SICS and dNaM. Chem. – Eur. J. 2017, 23, 11494– 11498, DOI: 10.1002/chem.20170258322https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1KqurbJ&md5=0ad08a3d291ccb9a7a9d23ea7091720bVisible-light-mediated excited state relaxation in semi-synthetic genetic alphabet: d5SICS and dNaMBhattacharyya, Kalishankar; Datta, AyanChemistry - A European Journal (2017), 23 (48), 11494-11498CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)The excited state dynamics of an unnatural base pair (UBP) d5SICS/dNaM were investigated by accurate ab-initio calcns. Time-dependent d. functional and high-level multireference calcns. (MS-CASPT2) were performed to elucidate the excitation of this UBP and its excited state relaxation mechanism. After excitation to the bright state S2 (ππ*), it decays to the S1 state and then undergoes efficient intersystem crossing to the triplet manifold. The presence of sulfur atom in d5SICS leads to strong spin-orbit coupling (SOC) and a small energy gap that facilitates intersystem crossing from S1 (nsπ*) to T2 (ππ*) followed by internal conversion to T1 state. Similarly in dNaM, the deactivation pathway follows analogous trends. CASPT2 calcns. suggest that the S1 (ππ*) state is a dark state below the accessible S2 (ππ*) bright state. During the ultrafast deactivation, it exhibits bond length inversion. From S1 state, significant SOC leads the population transfer to T3 due to a smaller energy gap. Henceforth, fast internal conversion occurs from T3 to T2 followed by T1. From time-dependent trajectory surface hopping dynamics, it is found that excited state relaxation occurs on a sub-picosecond timescale in d5SICS and dNaM. Our findings strongly suggest that there is enough energy available in triplet state of UBP to generate reactive oxygen species and induce phototoxicity with respect to cellular DNA.
- 23Pollum, M.; Ashwood, B.; Jockusch, S.; Lam, M.; Crespo-Hernández, C. E. Unintended Consequences of Expanding the Genetic Alphabet. J. Am. Chem. Soc. 2016, 138, 11457– 11460, DOI: 10.1021/jacs.6b0682223https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVWlsbbM&md5=73a69f7e204452afb85b39c530d95aecUnintended Consequences of Expanding the Genetic AlphabetPollum, Marvin; Ashwood, Brennan; Jockusch, Steffen; Lam, Minh; Crespo-Hernandez, Carlos E.Journal of the American Chemical Society (2016), 138 (36), 11457-11460CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The base pair d5SICS·dNaM was recently reported to incorporate and replicate in the DNA of a modified strain of Escherichia coli, thus making the world's first stable semisynthetic organism. This newly expanded genetic alphabet may allow organisms to store considerably more information in order to translate proteins with unprecedented enzymic activities. Importantly, however, there is currently no knowledge of the photochem. properties of d5SICS or dNaM-properties that are central to the chem. integrity of cellular DNA. In this contribution, it is shown that excitation of d5SICS or dNaM with near-visible light leads to efficient trapping of population in the nucleoside's excited triplet state in high yield. Photoactivation of these long-lived, reactive states is shown to photosensitize cells, leading to the generation of reactive oxygen species and to a marked decrease in cell proliferation, thus warning scientists of the potential phototoxic side effects of expanding the genetic alphabet.
- 24Feldmann, A. W.; Romesberg, F. E. Expansion of the genetic alphabet: A Chemist’s approach to synthetic biology. Acc. Chem. Res. 2018, 51, 394– 403, DOI: 10.1021/acs.accounts.7b00403There is no corresponding record for this reference.
- 25Lavergne, T.; Malyshev, D. A.; Romesberg, F. E. Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairs. Chem. – Eur. J. 2012, 18, 1231– 1239, DOI: 10.1002/chem.20110206625https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhs1Ghur3L&md5=e3fe084c25ab1596e07e721de72d4408Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairsLavergne, Thomas; Malyshev, Denis A.; Romesberg, Floyd E.Chemistry - A European Journal (2012), 18 (4), 1231-1239, S1231/1-S1231/27CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)Expansion of the genetic alphabet with an unnatural base pair is a long-standing goal of synthetic biol. The authors have developed a class of unnatural base pairs, formed between d5SICS and analogs of dMMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, the authors report the synthesis of five new dMMO2 analogs bearing different substituents designed to be oriented into the developing major groove and an anal. of their insertion opposite d5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the anal. of the previously optimized pair, dNaM-d5SICS, to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examd. The resulting structure-activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addn., the authors identify an analog (dNMO1) that is a better partner for d5SICS than any of the previously identified dMMO2 analogs with the exception of dNaM. They also found that dNaM-d5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.
- 26Dhami, K.; Malyshev, D. A.; Ordoukhanian, P.; Kubelka, T.; Hocek, M.; Romesberg, F. E. Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabet. Nucleic Acids Res. 2014, 42, 10235– 10244, DOI: 10.1093/nar/gku71526https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVCmtLnL&md5=4eefc0a675b6075fd06398d74400ad19Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabetDhami, Kirandeep; Malyshev, Denis A.; Ordoukhanian, Phillip; Kubelka, Tomas; Hocek, Michal; Romesberg, Floyd E.Nucleic Acids Research (2014), 42 (16), 10235-10244CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)We have developed a family of unnatural base pairs (UBPs), which rely on hydrophobic and packing interactions for pairing and which are well replicated and transcribed. While the pair formed between d5SICS and dNaM (d5SICS-dNaM) has received the most attention, and has been used to expand the genetic alphabet of a living organism, recent efforts have identified dTPT3-dNaM, which is replicated with even higher fidelity. These efforts also resulted in more UBPs than could be independently analyzed, and thus we now report a PCR-based screen to identify the most promising. While we found that dTPT3-dNaM is generally the most promising UBP, we identified several others that are replicated nearly as well and significantly better than d5SICS-dNaM, and are thus viable candidates for the expansion of the genetic alphabet of a living organism. Moreover, the results suggest that continued optimization should be possible, and that the putatively essential hydrogen-bond acceptor at the position ortho to the glycosidic linkage may not be required. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new structure-activity relationship data and importantly identify multiple new candidates for in vivo evaluation and further optimization.
- 27Lavergne, T.; Degardin, M.; Malyshev, D. A.; Quach, H. T.; Dhami, K.; Ordoukhanian, P.; Romesberg, F. E. Expanding the scope of replicable unnatural DNA: stepwise optimization of a predominantly hydrophobic base pair. J. Am. Chem. Soc. 2013, 135, 5408– 5419, DOI: 10.1021/ja312148q27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltV2msbY%253D&md5=0aa95cf5b916e8b619497821d808fe84Expanding the Scope of Replicable Unnatural DNA: Stepwise Optimization of a Predominantly Hydrophobic Base PairLavergne, Thomas; Degardin, Melissa; Malyshev, Denis A.; Quach, Henry T.; Dhami, Kirandeep; Ordoukhanian, Phillip; Romesberg, Floyd E.Journal of the American Chemical Society (2013), 135 (14), 5408-5419CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)As part of an ongoing effort to expand the genetic alphabet for in vitro and eventually in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs exemplified by d5SICS-dMMO2 and d5SICS-dNaM. When incorporated into DNA, the latter is replicated and transcribed with greater efficiency and fidelity than the former; however, previous optimization efforts identified the para and methoxy-distal meta positions of dMMO2 as particularly promising for further optimization. Here, we report the stepwise optimization of dMMO2 via the synthesis and evaluation of 18 novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most promising analogs with meta substituents. Subject to size constraints, we find that para substituents can optimize replication via both steric and electronic effects and that meta methoxy groups are unfavorable, while fluoro substituents can be beneficial or deleterious depending on the para substituent. In addn., we find that improvements in the efficiency of unnatural triphosphate insertion translate most directly into higher fidelity replication. Importantly, we identify multiple, unique base pair derivs. that when incorporated into DNA are well replicated. The most promising, d5SICS-dFEMO, is replicated under some conditions with greater efficiency and fidelity than d5SICS-dNaM. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new SAR data, and importantly identify multiple new candidates for eventual in vivo evaluation.
- 28Pollum, M.; Crespo-Hernández, C. E. Communication: the dark singlet state as a doorway state in the ultrafast and efficient intersystem crossing dynamics in 2-thiothymine and 2-thiouracil. J. Chem. Phys. 2014, 140, 071101 DOI: 10.1063/1.486644728https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjslKmt7w%253D&md5=666eb3d15e330fb467ad89e97b939214Communication: The dark singlet state as a doorway state in the ultrafast and efficient intersystem crossing dynamics in 2-thiothymine and 2-thiouracilPollum, Marvin; Crespo-Hernandez, Carlos E.Journal of Chemical Physics (2014), 140 (7), 071101/1-071101/5CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)Femtosecond broadband transient absorption expts. are reported for 2-thiothymine and 2-thiouracil in aq. buffer soln. and in acetonitrile. The S1(nπ*) state acts as a doorway state in the ultrafast and efficient population of the T1(ππ*) state upon 316 nm excitation. A sequential kinetic model is presented to explain the excited-state dynamics in 2-thiothymine and 2-thiouracil upon UVA excitation: S2(ππ*) → S1(nπ*) → T1(ππ*). The exptl. results are also used to scrutinize the excited-state relaxation pathways recently predicted for 2-thiouracil at the CASPT2//CASSCF level of theory. The efficient population of the T1(ππ*) state for both 2-thiothymine and 2-thiouracil in a few hundreds of femtoseconds lends further support to the emerging idea that thiobase derivs. exhibit photo-toxic properties that can be effectively harnessed in photo-chemotherapeutic applications. (c) 2014 American Institute of Physics.
- 29Pollum, M.; Jockusch, S.; Crespo-Hernández, C. E. 2,4-Dithiothymine as a potent UVA chemotherapeutic agent. J. Am. Chem. Soc. 2014, 136, 17930– 17933, DOI: 10.1021/ja510611j29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitFOgsLzO&md5=a8d7ac77d8ff134caf66b1d7df63671a2,4-Dithiothymine as a Potent UVA Chemotherapeutic AgentPollum, Marvin; Jockusch, Steffen; Crespo-Hernandez, Carlos E.Journal of the American Chemical Society (2014), 136 (52), 17930-17933CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Substitution of both oxygen atoms in the exocyclic carbonyl groups of the thymine chromophore by sulfur atoms results in a remarkable red shift of its absorption spectrum from an absorption max. at 267 nm in thymidine to 363 nm in 2,4-dithiothymine (ΔE = 9905 cm-1). A single sulfur substitution of a carbonyl group in the thymine chromophore at position 2 or 4 results in a significantly smaller red shift in the absorption max., which depends sensitively on the position at which the sulfur atom is substituted, varying from 275 nm in 2-thiothymine to 335 nm in 4-thiothymidine. Femtosecond transient absorption spectroscopy reveals that excitation of 2,4-dithiothymine at 335 or 360 nm leads to the ultrafast population of the triplet state, with an intersystem crossing lifetime of 180 ± 40 fs-the shortest intersystem crossing lifetime of any DNA base deriv. studied so far in aq. soln. Surprisingly, the degree and position at which the sulfur atom is substituted have important effects on the magnitude of the intersystem crossing rate const., showing a 1.2-, 3.2-, and 4.2-fold rate increases for 2-thiothymine, 4-thiothymidine, and 2,4-dithiothymine, resp., relative to that of thymidine, whereas the triplet yield increases 60-fold to near unity, independent of the site of sulfur atom substitution. While the natural thymine monomers owe their high degree of photostability to ultrafast internal conversion to the ground state and low triplet yields, the near-unity triplet yields in the thiothymine series account for their potent photosensitization properties. Nanosecond time-resolved luminescence spectroscopy shows that 4-thiothymidine and 2,4-dithiothymine are efficient singlet oxygen generators, with singlet oxygen quantum yields of 0.42 ± 0.02 and 0.46 ± 0.02, resp., in O2-satd. acetonitrile soln. Taken together, these photophys. measurements strongly suggest that 2,4-dithiothymine can act as a more effective UVA chemotherapeutic agent than the currently used 4-thiothymidine, esp. in deeper-tissue chemotherapeutic applications.
- 30Pollum, M.; Jockusch, S.; Crespo-Hernández, C. E. Increase in the photoreactivity of uracil derivatives by doubling thionation. Phys. Chem. Chem. Phys. 2015, 17, 27851– 27861, DOI: 10.1039/C5CP04822B30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFKhtbjN&md5=9931a8877b0f1e9b31fc665c6fb9ec3bIncrease in the photoreactivity of uracil derivatives by doubling thionationPollum, M.; Jockusch, S.; Crespo-Hernandez, C. E.Physical Chemistry Chemical Physics (2015), 17 (41), 27851-27861CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)The ability of 4-thiouracil to strongly absorb UVA radiation and to populate a reactive triplet state in high yield has enabled its use as a versatile photocrosslinker for nearly 50 years. In this contribution, we present a detailed spectroscopic and photochem. investigation of the 2-thiouracil, 4-thiouracil, and 2,4-dithiouracil series in an effort to further advance this chem. and to scrutinize the photoreactivity of 2,4-dithiouracil. Our results reveal that excitation of 2,4-dithiouracil leads to intersystem crossing to the triplet manifold in 220 ± 40 fs, which enables the population of the reactive triplet state with near unity yield (ΦT = 0.90 ± 0.15) and ultimately leads to a ca. 50% singlet oxygen generation (ΦΔ = 0.49 ± 0.02)-one of the highest singlet oxygen yields reported to date for a photoexcited thiobase. In addn., the long-lived triplet state of 2,4-dithiouracil reacts efficiently with the nucleic acid base adenine 5'-monophosphate through a direct, oxygen-independent photocycloaddn. mechanism and at a rate that is at least 3-fold faster than that of 4-thiouracil under equal conditions. The new physico-chem. insights reported for these RNA-thiobase derivs. are compared to those of the DNA and RNA bases and the DNA-thiobase derivs. Furthermore, the strong near-visible absorption and increased photoreactivity measured for 2,4-dithiouracil lays a solid foundation for developing RNA-targeted photocrosslinking and phototherapeutic agents that are more effective than those currently available.
- 31Dien, V. T.; Holcomb, M.; Feldman, A. W.; Fischer, E. C.; Dwyer, T. J.; Romesberg, F. E. Progress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic Alphabet. J. Am. Chem. Soc. 2018, 140, 16115– 16123, DOI: 10.1021/jacs.8b0841631https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFGmtrbP&md5=206c2634fd4753e9bdaafd9959f3023bProgress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic AlphabetDien, Vivian T.; Holcomb, Matthew; Feldman, Aaron W.; Fischer, Emil C.; Dwyer, Tammy J.; Romesberg, Floyd E.Journal of the American Chemical Society (2018), 140 (47), 16115-16123CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have developed a family of unnatural base pairs (UBPs), exemplified by the pair formed between dNaM and dTPT3, for which pairing is mediated not by complementary hydrogen bonding but by hydrophobic and packing forces. These UBPs enabled the creation of the first semisynthetic organisms (SSOs) that store increased genetic information and use it to produce proteins contg. noncanonical amino acids. However, retention of the UBPs was poor in some sequence contexts. Here, to optimize the SSO, we synthesize two novel benzothiophene-based dNaM analogs, dPTMO and dMTMO, and characterize the corresponding UBPs, dPTMO-dTPT3 and dMTMO-dTPT3. We demonstrate that these UBPs perform similarly to, or slightly worse than, dNaM-dTPT3in vitro. However, in the in vivo environment of an SSO, retention of dMTMO-dTPT3, and esp. dPTMO-dTPT3, is significantly higher than that of dNaM-dTPT3. This more optimal in vivo retention results from better replication, as opposed to more efficient import of the requisite unnatural nucleoside triphosphates. Modeling studies suggest that the more optimal replication results from specific internucleobase interactions mediated by the thiophene sulfur atoms. Finally, we show that dMTMO and dPTMO efficiently template the transcription of RNA contg. TPT3 and that their improved retention in DNA results in more efficient prodn. of proteins with noncanonical amino acids. This is the first instance of using performance within the SSO as part of the UBP evaluation and optimization process. From a general perspective, the results demonstrate the importance of evaluating synthetic biol. "parts" in their in vivo context and further demonstrate the ability of hydrophobic and packing interactions to replace the complementary hydrogen bonding that underlies the replication of natural base pairs. From a more practical perspective, the identification of dMTMO-dTPT3 and esp. dPTMO-dTPT3 represents significant progress toward the development of SSOs with an unrestricted ability to store and retrieve increased information.
- 32Jahiruddin, S.; Mandal, N.; Datta, A. Structure and Electronic Properties of Unnatural Base Pairs: The Role of Dispersion Interactions. ChemPhysChem 2018, 19, 67– 74, DOI: 10.1002/cphc.20170099732https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFGmtbjE&md5=e5af77161a0f2967c67bf044b889a9d9Structure and electronic properties of unnatural base pairs: The role of dispersion interactionsJahiruddin, Sk; Mandal, Nilangshu; Datta, AyanChemPhysChem (2018), 19 (1), 67-74CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)Recent reports of the successful incorporation of unnatural base pairs (UBPs), such as d5SICS-dNaM, in the gene sequence and replication with DNA is an important milestone in synthetic biol. Followed by this, several other UBPs, such as dTPT3-dNaM, dTPT3-dFIMO, dTPT3-IMO, dTPT3-FEMO, FTPT3-NaM, FTPT3-FIMO, FTPT3-IMO, and FTPT3-FEMO, have demonstrated similar or better retention and fidelity inside cells. Of these base pairs, dNaM-dTPT3 has been optimized to be a better fit inside a pAIO plasmid. Based on both implicit and explicit dispersion-cor. d. functional theory (DFT) calcns., we show that although this set of UBPs is significantly diverse in elemental and structural configuration, the members do share a common trait of favoring a slipped parallel stacked dimer arrangement. Unlike the natural bases (A, T, G, C, and U), this set of UBPs has a negligible affinity for a Watson-Crick (WC)-type planar structure because they are invariably more stable within slipped parallel stacked orientations. We also obsd. that all the UBPs have either similar or higher binding energies with the natural bases in similar stacked orientations. When arranged between two natural base pairs, the UBPs exhibited a binding energy similar to that of three-base sequences of natural bases. Our computational data show that the most promising base pairs are 5SICS-NaM, TPT3-NaM, and TPT3-FEMO. These results are consistent with recent progress on exptl. research into UBPs along with our previous calcns. on the d5SICS-dNaM pair and, therefore, strengthen the hypothesis that hydrogen bonding might not be absolutely essential and that interbase stacking dispersion interactions play a key role in the stabilization of genetic materials.
- 33Negi, I.; Kathuria, P.; Sharma, P.; Wetmore, S. D. How do hydrophobic nucleobases differ from natural DNA nucleobases? Comparison of structural features and duplex properties from QM calculations and MD simulations. Phys. Chem. Chem. Phys. 2017, 19, 16365– 16374, DOI: 10.1039/C7CP02576A33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpsVCmt7k%253D&md5=bdd66a0051cfd743e7cec50b48c75744How do hydrophobic nucleobases differ from natural DNA nucleobases? Comparison of structural features and duplex properties from QM calculations and MD simulationsNegi, Indu; Kathuria, Preetleen; Sharma, Purshotam; Wetmore, Stacey D.Physical Chemistry Chemical Physics (2017), 19 (25), 16365-16374CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)Computational (DFT and MD simulation) methods are employed to systematically characterize the structural and energetic properties of five hydrophobic nucleobases (FEMO, MMO2, NaM, 5SICS and TPT3) that constitute four unnatural base pairs (FEMO:5SICS, MMO2:5SICS, NaM:5SICS and TPT3:NaM). These hydrophobic bases have been recently shown to be replicated when present between natural bases in DNA duplexes, with the highest replication fidelity and efficiency occurring for the TPT3:NaM pair. Our QM calcns. suggest that the preferred (anti) glycosidic orientations of nucleosides contg. hydrophobic bases are similar to the natural DNA nucleosides despite differences in their chem. structures. However, due to the inability to form interbase hydrogen bonds, hydrophobic base pairs intrinsically prefer nonplanar, distorted geometries, many of which are stabilized through π-π stacking interactions. Furthermore, the intrinsic stacking potential between a hydrophobic and a natural base is similar to that between two natural bases, indicating that the strength of stacking interactions in DNA duplexes contg. hydrophobic bases is likely comparable to natural DNA. However, in contrast to the isolated base-pair geometries, our MD simulations suggest that the hydrophobic base pairs adopt variable geometries within DNA, which range from stacked (5SICS:FEMO) to nearly planar (5SICS:NaM and SICS:MMO2) to planar (TPT3:NaM). As a result, the duplex structural features at the site of modification depend on the identity of the hydrophobic base pair, where the TPT3:NaM pair causes the least structural changes compared to natural DNA. Overall, the structural insight obtained from our calcns. on DNA contg. hydrophobic base pairs explains the exptl.-obsd. higher fidelity and efficiency during replication of TPT3:NaM compared to other hydrophobic nucleobase pairs. By providing valuable structural information that explains the intrinsic and duplex properties of this class of unnatural nucleobases, the present work may aid the future design of improved hydrophobic analogs.
- 34Leconte, A. M.; Hwang, G. T.; Matsuda, S.; Capek, P.; Hari, Y.; Romesberg, F. E. Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet. J. Am. Chem. Soc. 2008, 130, 2336– 2343, DOI: 10.1021/ja078223d34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXps1KmtQ%253D%253D&md5=e8a92b3f3735fd4a116ca3a69534485bDiscovery, Characterization, and Optimization of an Unnatural Base Pair for Expansion of the Genetic AlphabetLeconte, Aaron M.; Hwang, Gil Tae; Matsuda, Shigeo; Capek, Petr; Hari, Yoshiyuki; Romesberg, Floyd E.Journal of the American Chemical Society (2008), 130 (7), 2336-2343CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)DNA is inherently limited by its four natural nucleotides. Efforts to expand the genetic alphabet, by addn. of an unnatural base pair, promise to expand the biotechnol. applications available for DNA as well as to be an essential first step toward expansion of the genetic code. We have conducted two independent screens of hydrophobic unnatural nucleotides to identify novel candidate base pairs that are well recognized by a natural DNA polymerase. From a pool of 3600 candidate base pairs, both screens identified the same base pair, dSICS:dMMO2, which we report here. Using a series of related analogs, we performed a detailed structure-activity relationship anal., which allowed us to identify the essential functional groups on each nucleobase. From the results of these studies, we designed an optimized base pair, d5SICS:dMMO2, which is efficiently and selectively synthesized by Kf within the context of natural DNA.
- 35Henry, A. A.; Yu, C.; Romesberg, F. E. Determinants of unnatural nucleobase stability and polymerase recognition. J. Am. Chem. Soc. 2003, 125, 9638– 9646, DOI: 10.1021/ja035398o35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXlsFOhsb4%253D&md5=f1e6a734e700d316fab4e25b738666f4Determinants of Unnatural Nucleobase Stability and Polymerase RecognitionHenry, Allison A.; Yu, Chengzhi; Romesberg, Floyd E.Journal of the American Chemical Society (2003), 125 (32), 9638-9646CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Six new unnatural nucleobases have been synthesized and characterized in terms of stability and selectivity of self-pairing in duplex DNA and efficiency and fidelity of self-pairing during polymerase-mediated replication. Each nucleobase has a conserved ring structure but differs from the others in its specific pattern of substitution with oxygen and sulfur atoms. Heteroatom derivatization within the conserved scaffold is shown to have only moderate effects on unnatural self-pair synthesis by the polymerase; larger effects were obsd. on the thermal stability and polymerase-mediated extension of the self-pairs. The largest effects of heteroatom substitution were on the stability and synthesis of mispairs between the unnatural and natural bases. Certain heteroatom substitutions were found to have a general effect while others were found to have effects that were specific for a particular unnatural or natural base. The data are useful for designing stable and replicable third base pairs and for understanding the contributions of nucleobase shape, polarity, and polarizability to the stability and replication of DNA.
- 36New, J. S.; Christopher, W. L.; Yevich, J. P.; Butler, R.; Schlemmer, R. F., Jr.; VanderMaelen, C. P.; Cipollina, J. A. The thieno[3,2-c]pyridine and furo[3,2-c]pyridine rings: new pharmacophores with potential antipsychotic activity. J. Med. Chem. 1989, 32, 1147– 1156, DOI: 10.1021/jm00126a00236https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXitlWmurw%253D&md5=098cfbe9523216238b07e82c1e01f0c4The thieno[3,2-c]pyridine and furo[3,2-c]pyridine rings: new pharmacophores with potential antipsychotic activityNew, James S.; Christopher, William L.; Yevich, Joseph P.; Butler, Rhett; Schlemmer, R. Francis, Jr.; VanderMaelen, Cam P.; Cipollina, Joseph A.Journal of Medicinal Chemistry (1989), 32 (6), 1147-56CODEN: JMCMAR; ISSN:0022-2623.Imidobutylpiperazinylpyrrolo-, -thieno-, and -furo[3,2-c]pyridines (e.g. I, X = O, Me2C; X1 = MeN, S, O) were synthesized. A no. of I (X1 = S, O) had significant activity in the blockade of apomorphine stereotypy and apomorphine-induced climbing, the Sidman avoidance response, and the conditioned avoidance response. In addn., while potent affinity for serotonin 5-HT2 and 5-HT2 receptors was obsd. for both the thieno- and furo[3,2-c]pyridine derivs., the interaction of these mols. with the dopamine D2 receptor was weak. Electrophysiol. studies of the lead prototypes I (X = Me2C, X1 = S; X = X1 = O), indicate these two mols. have distinctively different effects on dopamine neurons in areas A9 and A10. Despite the similarity these mols. share in their behavioral indexes of antipyschotic activity, it is likely that the thieno- and furo[3,2-c]pyridine rings employ different mechanisms to achieve this convergence of biol. effects.
- 37Asagarasu, A.; Matsui, T.; Hayashi, H.; Tamaoki, S.; Yamauchi, Y.; Sato, M. Design and Synthesis of Piperazinylpyridine Derivatives as Novel 5-HT1A Agonists/5-HT3 Antagonists for the Treatment of Irritable Bowel Syndrome (IBS). Chem. Pharm. Bull. 2009, 57, 34– 42, DOI: 10.1248/cpb.57.3437https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmsVGltr8%253D&md5=f075d0d527feed2dfd129199deb3d775Design and synthesis of piperazinylpyridine derivatives as novel 5-HT1A agonists/5-HT3 antagonists for the treatment of irritable bowel syndrome (IBS)Asagarasu, Akira; Matsui, Teruaki; Hayashi, Hiroyuki; Tamaoki, Satoru; Yamauchi, Yukinao; Sato, MichitakaChemical & Pharmaceutical Bulletin (2009), 57 (1), 34-42CODEN: CPBTAL; ISSN:0009-2363. (Pharmaceutical Society of Japan)We have prepd. a series of piperazinylpyridine derivs. for the treatment of irritable bowel syndrome (IBS). These compds., which were designed by pharmacophore anal., bind to both serotonin subtype 1A (5-HT1A) and subtype 3 (5-HT3) receptors. The nitrogen atom of the isoquinoline, a methoxy group and piperazine were essential to the pharmacophore for binding to these receptors. We also synthesized furo- and thienopyridine derivs. according to structure-activity relationship analyses. Compd. 17a (TZB-20810, I) had high affinities to these receptors and exhibited 5-HT1A agonistic activity and 5-HT3 antagonistic activity concurrently, and is a promising drug for further development in the treatment of IBS.
- 38Gueymard, C. A.; Myers, D.; Emery, K. Proposed reference irradiance spectra for solar energy systems testing. Solar Energy. 2002, 73, 443– 467, DOI: 10.1016/S0038-092X(03)00005-7There is no corresponding record for this reference.
- 39Morris, S. E.; Feldman, A. W.; Romesberg, F. E. Synthetic Biology Parts for the Storage of Increased Genetic Information in Cells. ACS Synth. Biol. 2017, 6, 1834– 1840, DOI: 10.1021/acssynbio.7b0011539https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFyhu7vL&md5=4ec70cf80be8c5412e48b4b72f5a464aSynthetic biology parts for the storage of increased genetic information in cellsMorris, Sydney E.; Feldman, Aaron W.; Romesberg, Floyd E.ACS Synthetic Biology (2017), 6 (10), 1834-1840CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)To bestow cells with novel forms and functions, the goal of synthetic biol., we have developed the unnatural nucleoside triphosphates dNaMTP and dTPT3TP, which form an unnatural base pair (UBP) and expand the genetic alphabet. While the UBP may be retained in the DNA of a living cell, its retention is sequence-dependent. We now report a steady-state kinetic characterization of the rate with which the Klenow fragment of E. coli DNA polymerase I synthesizes the UBP and its mispairs in a variety of sequence contexts. Correct UBP synthesis is as efficient as for a natural base pair, except in one sequence context, and in vitro performance is correlated with in vivo performance. The data elucidate the determinants of efficient UBP synthesis, show that the dNaM-dTPT3 UBP is the first generally recognized natural-like base pair, and importantly, demonstrate that dNaMTP and dTPT3TP are well optimized and standardized parts for the expansion of the genetic alphabet.
- 40Ouaray, Z.; Benner, S. A.; Georgiadis, M. M.; Richards, N. G. J. Building better polymerases: Engineering the replication of expanded genetic alphabets. J. Biol. Chem. 2020, 295, 17046– 17059, DOI: 10.1074/jbc.REV120.01374540https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXpvVWisw%253D%253D&md5=c27c40b83d9428d8e4898a9ebf880707Building better polymerases: engineering the replication of expanded genetic alphabetsOuaray, Zahra; Benner, Steven A.; Georgiadis, Millie M.; Richards, Nigel G. J.Journal of Biological Chemistry (2020), 295 (50), 17046-17059CODEN: JBCHA3; ISSN:1083-351X. (American Society for Biochemistry and Molecular Biology)A review. DNA polymerases are today used throughout scientific research, biotechnol., and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here esp., natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chem. and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymic, and mol. dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.
- 41Percze, K.; Meszaros, T. Analysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA Polymerases. ChemBioChem 2020, 21, 2939– 2944, DOI: 10.1002/cbic.20200023641https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlOju7rO&md5=d868026ed6003b8049e79ca55011cbdaAnalysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA PolymerasesPercze, Krisztina; Meszaros, TamasChemBioChem (2020), 21 (20), 2939-2944CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)One of the pivotal steps in aptamer selection is the amplification of target-specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in-depth study of using 5-indolyl-AA-dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo-) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water-in-oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high-throughput sequence anal. showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries.
- 42Hwang, G. T.; Romesberg, F. E. Unnatural substrate repertoire of A, B, and X family DNA polymerases. J. Am. Chem. Soc. 2008, 130, 14872– 14882, DOI: 10.1021/ja803833h42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1ehsbzJ&md5=453d01da1b8844dbd1a15674b9612bc7Unnatural Substrate Repertoire of A, B, and X Family DNA PolymerasesHwang, Gil Tae; Romesberg, Floyd E.Journal of the American Chemical Society (2008), 130 (44), 14872-14882CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)As part of an effort to develop unnatural base pairs that are stable and replicable in DNA, we examd. the ability of five different polymerases to replicate DNA contg. four different unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs. The unnatural pairs were developed based on intensive studies using the Klenow fragment of DNA polymerase I from E. coli (Kf) and found to be recognized to varying degrees. The five addnl. polymerases characterized here include family A polymerases from bacteriophage T7 and Thermus aquaticus, family B polymerases from Thermococcus litoralis and Thermococcus 9°N-7, and the family X polymerase, human polymerase β. While we find that some aspects of unnatural base pair recognition are conserved among the polymerases, for example, the pair formed between two d3FB nucleotides is typically well recognized, the detailed recognition of most of the unnatural base pairs is generally polymerase dependent. In contrast, we find that the pair formed between d5SICS and dMMO2 is generally well recognized by all of the polymerases examd., suggesting that the determinants of efficient and general recognition are contained within the geometric and electronic structure of these unnatural nucleobases themselves. The data suggest that while the d3FB:d3FB pair is sufficiently well recognized by several of the polymerases for in vitro applications, the d5SICS:dMMO2 heteropair is likely uniquely promising for in vivo use. T7-mediated replication is esp. noteworthy due to strong mispair discrimination.
- 43Jahiruddin, S.; Datta, A. What sustains the unnatural base pairs (UBPs) with no hydrogen bonds. J. Phys. Chem. B. 2015, 119, 5839– 5845, DOI: 10.1021/acs.jpcb.5b0329343https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmvVygtbY%253D&md5=6e8c0394bac5cf300680757f358746a1What Sustains the Unnatural Base Pairs (UBPs) with No Hydrogen BondsJahiruddin, Sk; Datta, AyanJournal of Physical Chemistry B (2015), 119 (18), 5839-5845CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)The recent report of a hydrophobic unnatural base pair (UBP), d5SICS-dNaM, which replicated in DNA PCR and also sustained and synthesized a plasmid inside E. coli genome by Romesberg and coworkers, is intriguing. Quantum chem. calcns. show that the UBPs prefer a slipped parallel configuration to facilitate weak dispersion interactions somewhat similar to the so-called π-stacking interaction. Nevertheless, within a natural DNA tract, classical mol. dynamics simulations show that the backbone and neighboring stacked bases together reorient the UBPs in natural base pair-like planar environment. Our computed structure with an av. end-end distance, dC1'-C1' = 10.7 Å for d5SICS-dNaM is in excellent agreement with available crystal structure (PDB ID: 3SV3, planar UBP with dC1'-C1'crystal = 11.0 Å). Quantum mech. calcns. for the UBP flanked by two natural base-pairs (A-T) on top and on bottom on equilibrated MD structure found large binding energy (ΔE = -74.0 kcal/mol). The present calcns. therefore establish the fact that the hydrophobic UBPs can be stabilized by dispersion interactions with other base pairs in the DNA tract even in the absence of any hydrogen bonding between the UBPs themselves.
- 44Betz, K.; Malyshev, D. A.; Lavergne, T.; Welte, W.; Diederichs, K.; Dwyer, T. J.; Ordoukhanian, P.; Romesberg, F. E.; Marx, A. KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry. Nat. Chem. Biol. 2012, 8, 612– 614, DOI: 10.1038/nchembio.96644https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnvVyrurw%253D&md5=3f53105607a419a1d1326125178d682dKlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometryBetz, Karin; Malyshev, Denis A.; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Dwyer, Tammy J.; Ordoukhanian, Phillip; Romesberg, Floyd E.; Marx, AndreasNature Chemical Biology (2012), 8 (7), 612-614CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Many candidate unnatural DNA base pairs have been developed, but some of the best-replicated pairs adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replication for one such pair, dNaM-d5SICS, and show that efficient replication results from the polymerase itself, inducing the required natural-like structure.
- 45Betz, K.; Malyshev, D. A.; Lavergne, T.; Welte, W.; Diederichs, K.; Romesberg, F. E.; Marx, A. Structural insights into DNA replication without hydrogen bonds. J. Am. Chem. Soc. 2013, 135, 18637– 18643, DOI: 10.1021/ja409609j45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVKjt7zL&md5=59ebd1ad03d32be53c857c76ebf3a2c0Structural insights into DNA replication without hydrogen bondsBetz, Karin; Malyshev, Denis A.; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Romesberg, Floyd E.; Marx, AndreasJournal of the American Chemical Society (2013), 135 (49), 18637-18643CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The genetic alphabet is composed of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chem. potential of DNA. D5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq DNA polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson-Crick-like structure. To understand the remaining steps of replication, the authors now report the characterization of the prechem. complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple postchem. complexes in which the already formed unnatural base pair is positioned at the postinsertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addn. of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the postinsertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are obsd., depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by deintercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.
- 46Hirao, I.; Mitsui, T.; Kimoto, M.; Yokoyama, S. An efficient unnatural base pair for PCR amplification. J. Am. Chem. Soc. 2007, 129, 15549– 15555, DOI: 10.1021/ja073830m46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlejsbnO&md5=aa94eef33b0951eac30e79775e4623adAn Efficient Unnatural Base Pair for PCR AmplificationHirao, Ichiro; Mitsui, Tsuneo; Kimoto, Michiko; Yokoyama, ShigeyukiJournal of the American Chemical Society (2007), 129 (50), 15549-15555CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Expansion of the genetic alphabet by an unnatural base pair system provides a powerful tool for modern biotechnol. As an alternative to previous unnatural base pairs, we have developed a new pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitropyrrole (Pn), which functions in DNA amplification. Pn more selectively pairs with Ds in replication than another previously reported pairing partner, pyrrole-2-carbaldehyde (Pa). The nitro group of Pn efficiently prevented the mispairing with A. High efficiency and selectivity of the Ds-Pn pair in PCR amplification were achieved by using a substrate mixt. of the γ-amidotriphosphate of Ds and the usual triphosphates of Pn and the natural bases, with Vent DNA polymerase as a 3' to 5' exonuclease-proficient polymerase. After 20 cycles of PCR, the total mutation rate of the Ds-Pn site in an amplified DNA fragment was ∼1%. PCR amplification of DNA fragments contg. the unnatural Ds-Pn pair would be useful for expanded genetic systems in DNA-based biotechnol.
- 47Hashimoto, K.; Fischer, E. C.; Romesberg, F. E. Efforts toward Further Integration of an Unnatural Base Pair into the Biology of a Semisynthetic Organism. J. Am. Chem. Soc. 2021, 143, 8603– 8607, DOI: 10.1021/jacs.1c0386047https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXht1ejtbfM&md5=b730f6df8c45a65f5473e661030cf3b4Efforts toward Further Integration of an Unnatural Base Pair into the Biology of a Semisynthetic OrganismHashimoto, Koji; Fischer, Emil C.; Romesberg, Floyd E.Journal of the American Chemical Society (2021), 143 (23), 8603-8607CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)We have developed semisynthetic organisms (SSOs) that by virtue of a family of synthetic, unnatural base pairs (UBPs), store and retrieve increased information. To date, transcription in the SSOs has relied on heterologous expression of the RNA polymerase from T7 bacteriophage; here, we explore placing transcription under the control of the endogenous host multisubunit RNA polymerase. The results demonstrate that the E. coli RNA polymerase is able to transcribe DNA contg. a UBP and that with the most optimal UBP identified to date it should be possible to select for increased uptake of unnatural triphosphates. These advances should facilitate the creation of next generation SSOs.
- 48Zhang, Y.; Lamb, B. M.; Feldman, A. W.; Zhou, A. X.; Lavergne, T.; Li, L.; Romesberg, F. E. A semisynthetic organism engineered for the stable expansion of the genetic alphabet. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 1317– 1322, DOI: 10.1073/pnas.161644311448https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlCqurs%253D&md5=39e6053994d8c1a47d01833f515c18b0A semisynthetic organism engineered for the stable expansion of the genetic alphabetZhang, Yorke; Lamb, Brian M.; Feldman, Aaron W.; Zhou, Anne Xiaozhou; Lavergne, Thomas; Li, Lingjun; Romesberg, Floyd E.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (6), 1317-1322CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)All natural organisms store genetic information in a four-letter, two-base-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escherichia coli grown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA contg. a single dNaM-d5SICS UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used a more chem. optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet.
- 49Feldman, A. W.; Romesberg, F. E. In vivo structure-activity relationships and optimization of an unnatural base pair for replication in a semi-synthetic organism. J. Am. Chem. Soc. 2017, 139, 11427– 11433, DOI: 10.1021/jacs.7b0354049https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlShs7fE&md5=f86f88442184302b1777b217ea25c0abIn Vivo Structure-Activity Relationships and Optimization of an Unnatural Base Pair for Replication in a Semi-Synthetic OrganismFeldman, Aaron W.; Romesberg, Floyd E.Journal of the American Chemical Society (2017), 139 (33), 11427-11433CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)In an effort to expand the genetic alphabet and create semi-synthetic organisms (SSOs) that store and retrieve increased information, we have developed the unnatural base pairs (UBPs) dNaM and d5SICS or dTPT3 (dNaM-d5SICS and dNaM-dTPT3). The UBPs form based on hydrophobic and packing forces, as opposed to complementary hydrogen bonding, and while they are both retained within the in vivo environment of an Escherichia coli SSO, their development was based on structure-activity relationship (SAR) data generated in vitro. To address the likely possibility of different requirements of the in vivo environment, we screened 135 candidate UBPs for optimal performance in the SSO. Interestingly, we find that in vivo SARs differ from those collected in vitro, and most importantly, we identify four UBPs whose retention in the DNA of the SSO is higher than that of dNaM-dTPT3, which was previously the most promising UBP identified. The identification of these four UBPs further demonstrates that when optimized, hydrophobic and packing forces may be used to replace the complementary hydrogen bonding used by natural pairs and represents a significant advance in our continuing efforts to develop SSOs that store and retrieve more information than natural organisms.
- 50Seo, Y. J.; Hwang, G. T.; Ordoukhanian, P.; Romesberg, F. E. Optimization of an unnatural base pair toward natural-like replication. J. Am. Chem. Soc. 2009, 131, 3246– 3252, DOI: 10.1021/ja807853m50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXit1eksbc%253D&md5=0896876c67103ffd1845f2313624bd8bOptimization of an Unnatural Base Pair toward Natural-Like ReplicationSeo, Young Jun; Hwang, Gil Tae; Ordoukhanian, Phillip; Romesberg, Floyd E.Journal of the American Chemical Society (2009), 131 (9), 3246-3252CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Predominantly hydrophobic unnatural nucleotides that selectively pair within duplex DNA as well as during polymerase-mediated replication have recently received much attention as the cornerstone of efforts to expand the genetic alphabet. We recently reported the results of a screen and subsequent lead hit optimization that led to identification of the unnatural base pair formed between the nucleotides dMMO2 and d5SICS. This unnatural base pair is replicated by the Klenow fragment of Escherichia coli DNA polymerase I with better efficiency and fidelity than other candidates reported in the literature. However, its replication remains significantly less efficient than a natural base pair, and further optimization is necessary for its practical use. To better understand and optimize the slowest step of replication of the unnatural base pair, the insertion of dMMO2 opposite d5SICS, we synthesized two dMMO2 derivs., d5FM and dNaM, which differ from the parent nucleobase in terms of shape, hydrophobicity, and polarizability. We find that both derivs. are inserted opposite d5SICS more efficiently than dMMO2 and that overall the corresponding unnatural base pairs are generally replicated with higher efficiency and fidelity than the pair between dMMO2 and d5SICS. In fact, in the case of the dNaM:d5SICS heteropair, the efficiency of each individual step of replication approaches that of a natural base pair, and the min. overall fidelity ranges from 103 to 104. In addn., the data allow us to propose a generalized model of unnatural base pair replication, which should aid in further optimization of the unnatural base pair and possibly in the design of addnl. unnatural base pairs that are replicated with truly natural-like efficiency and fidelity.
- 51Malyshev, D. A.; Dhami, K.; Quach, H. T.; Lavergne, T.; Ordoukhanian, P.; Torkamani, A.; Romesberg, F. E. Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 12005– 12010, DOI: 10.1073/pnas.120517610951https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xht1GrtL3M&md5=a3e02f5c8ccda2071120e01ce1b059d1Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabetMalyshev, Denis A.; Dhami, Kirandeep; Quach, Henry T.; Lavergne, Thomas; Ordoukhanian, Phillip; Torkamani, Ali; Romesberg, Floyd E.Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (30), 12005-12010, S12005/1-S12005/31CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chem. and synthetic biol. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA contg. one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under std. conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates contg. the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that for PCR and PCR-based applications, d5SICS-dNaM is functionally equiv. to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.
- 52Sharma, P.; Manderville, R. A.; Wetmore, S. D. Structural and energetic characterization of the major DNA adduct formed from the food mutagen ochratoxin A in the NarI hotspot sequence: influence of adduct ionization on the conformational preferences and implications for the NER propensity. Mutagenic. The Significance of Internal Communication in the Management of Successful Change. Nucleic Acids Res. 2014, 42, 11831– 11845, DOI: 10.1093/nar/gku82152https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVChtr%252FF&md5=b92a626995c4d1f98cfaa6b77a58c787Structural and energetic characterization of the major DNA adduct formed from the food mutagen ochratoxin A in the NarI hotspot sequence: influence of adduct ionization on the conformational preferences and implications for the NER propensitySharma, Purshotam; Manderville, Richard A.; Wetmore, Stacey D.Nucleic Acids Research (2014), 42 (18), 11831-11845CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)The nephrotoxic food mutagen ochratoxin A (OTA) produces DNA adducts in rat kidneys, the major lesion being the C8-linked-2'-deoxyguanosine adduct (OTB-dG). Although research on other adducts stresses the importance of understanding the structure of the assocd. adducted DNA, site-specific incorporation of OTB-dG into DNA has yet to be attempted. The present work uses a robust computational approach to det. the conformational preferences of OTB-dG in three ionization states at three guanine positions in the NarI recognition sequence opposite cytosine. Representative adducted DNA helixes were derived from over 2160 ns of simulation and ranked via free energies. For the first time, a close energetic sepn. between three distinct conformations is highlighted, which indicates OTA-adducted DNA likely adopts a mixt. of conformations regardless of the sequence context. Nevertheless, the preferred conformation depends on the flanking bases and ionization state due to deviations in discrete local interactions at the lesion site. The structural characteristics of the lesion thus discerned have profound implications regarding its repair propensity and mutagenic outcomes, and support recent expts. suggesting the induction of double-strand breaks and deletion mutations upon OTA exposure. This combined structural and energetic characterization of the OTB-dG lesion in DNA will encourage future biochem. expts. on this potentially genotoxic lesion.
- 53Flamme, M.; Röthlisberger, P.; Levi-Acobas, F.; Chawla, M.; Oliva, R.; Cavallo, L.; Gasser, G.; Marlière, P.; Herdewijn, P.; Hollenstein, M. Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside Triphosphate. ACS Chem. Biol. 2020, 15, 2872– 2884, DOI: 10.1021/acschembio.0c0039653https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFagtLjK&md5=98c9d3978e55cb62ddb9db007684bf29Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside TriphosphateFlamme, Marie; Rothlisberger, Pascal; Levi-Acobas, Fabienne; Chawla, Mohit; Oliva, Romina; Cavallo, Luigi; Gasser, Gilles; Marliere, Philippe; Herdewijn, Piet; Hollenstein, MarcelACS Chemical Biology (2020), 15 (11), 2872-2884CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)The expansion of the genetic alphabet with addnl., unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biol. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymic construction of a silver-mediated base pair (dImC-AgI-dPurP) as well as a two-step protocol for the synthesis of DNA duplexes contg. such an artificial metal base pair. Guided by DFT calcns., we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymic synthesis of the dImC-AgI-dPurP artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.
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