这是用户在 2024-9-19 11:47 为 https://pubs.acs.org/doi/10.1021/jacs.7b11404 保存的双语快照页面，由 沉浸式翻译 提供双语支持。了解如何保存？

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect
ACS Publications. Most Trusted. Most Cited. Most Read
A Tool for the Import of Natural and Unnatural Nucleoside Triphosphates into Bacteria
More

OR SEARCH CITATIONS

My Activity
Publications
Recently Viewed
CONTENT TYPES

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Am. Chem. Soc. 2018, 140, 4, 1447-1454
  • Subscribed
Article

A Tool for the Import of Natural and Unnatural Nucleoside Triphosphates into Bacteria
将天然和非天然核苷三磷酸盐导入细菌的工具
Click to copy article link
点击复制文章链接Article link copied!

View Author Information
† Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
‡ Department of Pharmaceutical Sciences, University of California, Irvine, California 92697, United States
*floyd@scripps.edu
Open PDFSupporting Information (1)

Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2018, 140, 4, 1447–1454
Click to copy citationCitation copied!
https://doi.org/10.1021/jacs.7b11404
Published January 17, 2018
Copyright © 2018 American Chemical Society
Request reuse permissions

Abstract 抽象

Click to copy section link
单击以复制部分链接Section link copied!
High Resolution Image
Download MS PowerPoint Slide

Nucleoside triphosphates play a central role in biology, but efforts to study these roles have proven difficult because the levels of triphosphates are tightly regulated in a cell and because individual triphosphates can be difficult to label or modify. In addition, many synthetic biology efforts are focused on the development of unnatural nucleoside triphosphates that perform specific functions in the cellular environment. In general, both of these efforts would be facilitated by a general means to directly introduce desired triphosphates into cells. Previously, we demonstrated that recombinant expression of a nucleoside triphosphate transporter from Phaeodactylum tricornutum (PtNTT2) in Escherichia coli functions to import triphosphates that are added to the media. Here, to explore the generality and utility of this approach, we report a structure–activity relationship study of PtNTT2. Using a conventional competitive uptake inhibition assay, we characterize the effects of nucleobase, sugar, and triphosphate modification, and then develop an LC-MS/MS assay to directly measure the effects of the modifications on import. Lastly, we use the transporter to import radiolabeled or 2′-fluoro-modified triphosphates and quantify their incorporation into DNA and RNA. The results demonstrate the general utility of the PtNTT2-mediated import of natural or modified nucleoside triphosphates for different molecular or synthetic biology applications.
核苷三磷酸盐在生物学中起着核心作用，但事实证明，研究这些作用的努力是困难的，因为三磷酸盐的水平在细胞中受到严格调节，而且单个三磷酸盐可能难以标记或修饰。此外，许多合成生物学工作都集中在开发在细胞环境中执行特定功能的非天然核苷三磷酸盐。一般来说，这两种努力都可以通过直接将所需的三磷酸盐引入细胞的一般方法来促进。以前，我们证明了来自大肠杆菌 Phaeodactylum tricornutum （PtNTT2） 的核苷三磷酸转运蛋白的重组表达具有输入添加到培养基中的三磷酸盐的作用。在这里，为了探索这种方法的通用性和实用性，我们报告了 PtNTT2 的结构-活性关系研究。使用常规的竞争性摄取抑制测定法，我们表征了核碱基、糖和三磷酸盐修饰的影响，然后开发了 LC-MS/MS 测定法来直接测量修饰对输入的影响。最后，我们使用转运蛋白导入放射性标记或 2'-氟修饰的三磷酸盐，并定量它们掺入 DNA 和 RNA。结果表明，PtNTT2 介导的天然或修饰的核苷三磷酸输入在不同分子或合成生物学应用中的一般效用。

This publication is licensed under the terms of your institutional subscription. Request reuse permissions.
本出版物根据您的机构订阅条款获得许可。请求重用权限。

Copyright © 2018 American Chemical Society
版权所有 © 2018 美国化学会

Subjects

what are subjects

Introduction 介绍

Click to copy section link
单击以复制部分链接Section link copied!
Nucleoside triphosphates play a central role in virtually all aspects of biology. In addition to their fundamental roles as substrates for the synthesis of DNA and RNA and as the currency of cellular energy, nucleoside triphosphates can regulate transcription, translation, and inter- or intracellular signaling, and they are also required for glycogen, lipid, and cofactor synthesis. Unfortunately, characterizing the roles of nucleoside triphosphates in the cell is complicated by our inability to selectively label and/or experimentally control specific triphosphate concentrations. In addition, many recent efforts in synthetic biology have focused on the use of sugar- or nucleobase-modified triphosphates, for example, for the creation of novel biopolymers (1, 2) or the expansion of the genetic alphabet, (3-5) and ultimately all of these efforts require the availability of unnatural nucleoside triphosphates within the cell.
核苷三磷酸在生物学的几乎所有方面都起着核心作用。除了作为 DNA 和 RNA 合成的底物以及作为细胞能量货币的基本作用外，核苷三磷酸还可以调节转录、翻译和细胞间或细胞内信号传导，它们也是糖原、脂质和辅因子合成所必需的。不幸的是，由于我们无法选择性标记和/或实验控制特定的三磷酸盐浓度，因此表征三磷酸核苷在细胞中的作用变得复杂。此外，合成生物学领域的许多最新工作都集中在使用糖或核碱基修饰的三磷酸盐上，例如，用于创造新型生物聚合物 （1， 2） 或扩展遗传字母表 （3-5），最终所有这些努力都需要细胞内非天然核苷三磷酸盐的可用性。
One possible route to the introduction of natural or unnatural triphosphates into cells is the passive diffusion or facilitated uptake of the corresponding nucleosides across the cell membrane(s), (6-9) followed by their phosphorylation via the kinases of the nucleoside salvage pathway. (10) Indeed, this strategy is employed by many nucleoside prodrugs, such as azidothymidine (AZT) and gemcitabine, which are active as triphosphates. However, this approach is unlikely to be general, because many organisms, including fungi and bacteria, disproportionately or exclusively rely on de novo synthesis, and have lost some or all of the salvage kinases. (11-14) Furthermore, even if salvage kinases are present, their recognition of unnatural analogues may not be sufficient. (15) For example, gemcitabine is inactive against many Gram-negative bacteria due to poor conversion of the free nucleoside to the monophosphate, while AZT is inactive against many Gram-positive bacteria, at least in part, due to poor conversion of the monophosphate to the diphosphate. (16) In principle, these challenges may be overcome via heterologous expression of kinases with lower specificity. (17-21) However, the addition of exogenous kinase activity may perturb the balance of natural triphosphates within the cell, which is known to be toxic. (22-24) Regardless, whether with native or engineered pathways, relying on the activation of free nucleosides to produce the desired triphosphates is less than optimal, because it requires three steps of activation, and activation must compete with nucleoside degradation, because both eukaryotes and prokaryotes utilize nucleosides as sources of carbon and nitrogen. (25-27) This is likely to increase the challenge of achieving controlled intracellular concentrations of the triphosphates, which is likely to be problematic with many applications.
将天然或非天然三磷酸盐引入细胞的一种可能途径是相应核苷在细胞膜上的被动扩散或促进摄取，（6-9），然后通过核苷补救途径的激酶磷酸化。（10） 事实上，许多核苷前药都采用了这种策略，例如叠氮胸苷 （AZT） 和吉西他滨，它们以三磷酸盐的形式具有活性。然而，这种方法不太可能是通用的，因为许多生物体，包括真菌和细菌，不成比例或完全依赖于从头合成，并且已经失去了部分或全部补救激酶。（11-14） 此外，即使存在挽救激酶，它们对非自然类似物的识别也可能不够。（15） 例如，吉西他滨由于游离核苷转化为单磷酸盐的转化不良，对许多革兰氏阴性菌无活性，而 AZT 对许多革兰氏阳性菌无活性，至少部分是由于单磷酸盐向二磷酸盐的转化不良。（16） 原则上，这些挑战可以通过特异性较低的激酶的异源表达来克服。（17-21） 然而，外源激酶活性的添加可能会扰乱细胞内天然三磷酸盐的平衡，这是已知的有毒的。（22-24） 无论如何，无论是天然途径还是工程途径，依靠游离核苷的激活来产生所需的三磷酸盐都不是最佳的，因为它需要三个激活步骤，并且激活必须与核苷降解竞争，因为真核生物和原核生物都利用核苷作为碳和氮的来源。 （25-27） 这可能会增加实现三磷酸盐细胞内受控浓度的挑战，这在许多应用中可能存在问题。
As part of an effort to create semisynthetic organisms that by virtue of an unnatural base pair store (24, 28) and retrieve (29) increased genetic information, we have reported that the constituent unnatural nucleoside triphosphates, which bear nucleobases with little homology to their natural counterparts, are imported directly into Escherichia coli through a heterologously expressed nucleoside triphosphate transporter (NTT) from Phaeodactylum tricornutum (PtNTT2). (30) In its native algae, PtNTT2 is thought to mediate the counter exchange of nucleoside triphosphates across the outer plastidial membrane from the cytoplasm to the stroma, where they are required for DNA and RNA synthesis. When expressed in E. coli, PtNTT2 is inserted into the cytoplasmic membrane, where it transports nucleoside triphosphates into the cytoplasm after they first diffuse from the media into the periplasm, presumably through porins. While PtNTT2 is selective for triphosphates over other nucleotides, its ability to transport all eight deoxy- and ribonucleoside triphosphates endows it with the broadest substrate scope of any known NTT, although the different natural substrates are transported with somewhat different efficiencies. Further, our observation that PtNTT2 may be used to import triphosphates with wholly unnatural nucleobases indicates that the substrate scope of PtNTT2 may extend far beyond the natural nucleotides. This suggests that PtNTT2 may be a useful tool for a variety of molecular and synthetic biology applications requiring the availability of unnatural nucleoside triphosphates within a cell.
作为创造半合成生物的努力的一部分，这些生物体通过非天然碱基对存储 （24， 28） 和检索 （29） 增加的遗传信息，我们已经报道了组成非天然核苷三磷酸盐，其核碱基与其天然对应物几乎没有同源性，通过异源表达的核苷三磷酸转运蛋白 （NTT） 直接输入到大肠杆菌中 Phaeodactylum tricornutum （第NTT2 部分）。（30） 在其天然藻类中，PtNTT2 被认为介导三磷酸核苷跨外质膜从细胞质到基质的反交换，它们是 DNA 和 RNA 合成所必需的。当在大肠杆菌中表达时，PtNTT2入细胞质膜，在三磷酸核苷首先从培养基扩散到周质中后，它可能通过孔蛋白将三磷酸核苷转运到细胞质中。虽然 PtNTT2 对三磷酸盐的选择性优于其他核苷酸，但它运输所有八种脱氧和核糖核苷三磷酸盐的能力使其具有任何已知 NTT 中最广泛的底物范围，尽管不同的天然底物的运输效率略有不同。此外，我们观察到 PtNTT2 可用于输入具有完全非天然核碱基的三磷酸盐，这表明 PtNTT2 的底物范围可能远远超出天然核苷酸。这表明 PtNTT2 可能是各种分子和合成生物学应用的有用工具，这些应用需要细胞内非天然的三磷酸核苷。
The standard assay used to characterize triphosphate uptake with PtNTT2, as well as other NTTs, is based on the use of radiolabeled substrates. (30) However, radiolabeled versions of many triphosphates of interest are not commercially available and are challenging to synthesize. To increase the range of substrates that may be examined, inhibition of the uptake of a commercially available radiolabeled substrate has also been used. (30) However, this assay by definition only characterizes the ability of the triphosphate of interest to competitively bind the NTT and does not actually characterize its uptake. Here, as part of an effort to develop NTTs as general tools for molecular and synthetic biology, we report a structure–activity relationship (SAR) study of PtNTT2 using the standard inhibition assay, to characterize transporter binding, and we develop a more general LC-MS/MS assay to characterize uptake directly, including the uptake of interesting unnatural triphosphates whose characterization is not possible with traditional assays. The data constitute the first SAR study of NTT activity and clarify the scope of analogues that might be made available in vivo for molecular and synthetic biology applications. Finally, we demonstrate the utility of PtNTT2 for the characterization of DNA replication and transcription in E. coli with both natural and modified nucleotides.
用于表征 PtNTT2 以及其他 NTT 对三磷酸盐摄取的标准测定基于放射性标记底物的使用。（30） 然而，许多感兴趣的三磷酸盐的放射性标记版本没有商业上可买到，并且难以合成。为了增加可以检查的底物范围，还使用了抑制市售放射性标记底物的摄取。（30） 然而，根据定义，该测定仅表征了感兴趣的三磷酸盐竞争性结合 NTT 的能力，实际上并没有表征其摄取。在这里，作为将 NTT 开发为分子和合成生物学通用工具的努力的一部分，我们报告了使用标准抑制测定法对 PtNTT2 进行的结构-活性关系 （SAR） 研究，以表征转运蛋白结合，并且我们开发了一种更通用的 LC-MS/MS 测定法来直接表征摄取，包括有趣的非天然三磷酸盐的摄取，其表征传统测定法无法实现。这些数据构成了 NTT 活性的首次 SAR 研究，并阐明了可能在体内用于分子和合成生物学应用的类似物的范围。最后，我们证明了 PtNTT2 在用天然和修饰核苷酸表征大肠杆菌中 DNA 复制和转录的效用。

Results and Discussion 结果与讨论

Click to copy section link
单击以复制部分链接Section link copied!
As with many membrane proteins, expression of native PtNTT2 is somewhat toxic in E. coli. However, we recently reported that removal of the protein’s N-terminal residues 1–65, which are normally removed in its native host, resulting in the variant PtNTT2(66–575), largely eliminates toxicity while preserving at least some level of activity. (28) This allowed us to construct E. coli YZ2, (28) which has PtNTT2(66–575) integrated into the chromosome under the control of the constitutive lacUV5 promoter. Chromosomal expression of PtNTT2(66–575) reduces variation in transporter expression, and presumably in uptake, compared to plasmid-based expression, due to reduced fluctuation in gene copy number. However, it is possible that deletion of the N-terminus alters activity. Thus, we first characterized the ability of the truncated transporter to import [α32P]ATP, using the standard radioactivity assay. Control experiments employing the BL21(DE3) CmR strain, which bears a chloramphenicol resistance marker at the locus that would otherwise carry the transporter, showed no detectable uptake, while YZ2 showed robust uptake. To characterize activity we determined normalized initial velocities of triphosphate uptake (expressed in the units fmol·cell–1·hr–1), and then plotted them against the concentration of [α32P]ATP added. Fitting the resulting curve to the Michaelis–Menten equation yielded the apparent KM and Vmax values, 186 ± 21.1 μM and 1.74 ± 0.091 × 10–2 fmol·cell–1·hr–1, respectively. Although we cannot compare Vmax as the absolute amount of transporter produced in strain YZ2 is unknown, the KM is in good agreement with that reported previously for the intact transporter, (30) indicating that the truncation does not significantly alter activity.
与许多膜蛋白一样，天然 PtNTT2 的表达在大肠杆菌中具有一定的毒性。然而，我们最近报道了去除蛋白质的 N 末端残基 1-65，这些残基通常在其天然宿主中被去除，导致变体 PtNTT2（66-575），在很大程度上消除了毒性，同时至少保留了一定程度的活性。（28） 这使我们能够构建大肠杆菌 YZ2，（28） 它在组成型 lacUV5 启动子的控制下将 PtNTT2（66–575） 整合到染色体中。与基于质粒的表达相比，PtNTT2 （66–575） 的染色体表达减少了转运蛋白表达的变异，并且可能减少了摄取，因为基因拷贝数的波动减少了。然而，N 末端的缺失可能会改变活性。因此，我们首先使用标准放射性测定表征了截短转运蛋白输入 [α32P] ATP 的能力。采用 BL21（DE3） CmR 菌株的对照实验，该菌株在基因座上带有氯霉素抗性标记，否则将携带转运蛋白，显示未检测到的摄取，而 YZ2 显示出强劲的摄取。为了表征活性，我们确定了三磷酸盐摄取的归一化初始速度（以单位 fmol·cell–1·hr–1 表示），然后将它们与添加的 [α32P] ATP 的浓度作图。将所得曲线拟合到 Michaelis-Menten 方程中，得到表观 KM 和 V最大值，分别为 186 ± 21.1 μM 和 1.74 ± 0.091 × 10-2 fmol·cell–1·hr–1。 虽然我们不能比较 Vmax，因为菌株 YZ2 中产生的转运蛋白的绝对量是未知的，但 KM 与之前报道的完整转运蛋白的 K M 非常一致，（30） 表明截断不会显着改变活性。

Analysis of Inhibition 抑制分析

To elucidate the SARs governing nucleoside triphosphate binding, we first employed the standard assay based on the inhibition of [α32P]ATP uptake. (30) Briefly, YZ2 cells were grown to an OD600 of ∼0.3 in 2×YT media, after which [α32P]ATP (50 μM) and a 10-fold excess of the triphosphate of interest were added. The cells were incubated for 10 min, during which time degradation due to extracellular phosphatases is insignificant, (24) and then transferred by vacuum to filter paper, which was washed to remove excess triphosphate and subjected to phosphorimaging to determine [α32P]ATP uptake. We initially focused on characterizing the SARs associated with the nucleobase and specifically on CTP with C5-modifications (Figure 1), because CTP is the most efficiently imported natural substrate, (30) and because such modifications are important in nature. (31-33) Both 5-iodo-2′-cytidine triphosphate (5-ICTP) and 5-iodo-2′-deoxycytidine triphosphate (5-IdCTP) potently inhibited [α32P]ATP uptake (96.6 ± 1.3% and 73.6 ± 1.8% inhibition, respectively). In fact, both of these analogues inhibited uptake more than or as much as their respective natural counterparts (70.5 ± 4.9% and 76.3 ± 2.8%, respectively). 5-hydroxymethyl cytidine (5-hmCTP) and 5-allyl amine cytidine (5-aaCTP) also potently inhibited uptake, but slightly less efficiently than their iodinated counterpart (76.3 ± 2.9% and 64.7 ± 9.3% inhibition, respectively). Based on this competitive inhibition data, it appears that the transporter is relatively tolerant of substitution at the C5-position of pyrimidine, with iodo substitution actually increasing inhibition.
为了阐明控制三磷酸核苷结合的 SAR，我们首先采用了基于抑制 [α32P] ATP 摄取的标准测定。（30） 简而言之，YZ2 细胞在 2×YT 培养基中生长至 ∼0.3 的 OD600，然后加入 [α32P]ATP （50 μM） 和 10 倍过量的目标三磷酸盐。将细胞孵育 10 分钟，在此期间，由于细胞外磷酸酶引起的降解微不足道，（24） 然后通过真空转移到滤纸上，过滤纸经过洗涤以去除多余的三磷酸盐，并进行磷光成像以确定 [α32P] ATP 摄取。我们最初专注于表征与核碱基相关的 SAR，特别是具有 C5 修饰的 CTP（图 1），因为 CTP 是最有效的输入天然底物 （30），并且这种修饰在自然界中很重要。（31-33） 5-碘-2′-胞苷三磷酸 （5-ICTP） 和 5-碘-2′-脱氧胞苷三磷酸 （5-IdCTP） 均有效抑制 [α 32P]ATP 摄取（分别为 96.6 ± 1.3% 和 73.6 ± 1.8%）。事实上，这两种类似物对摄取的抑制程度超过或等于各自的天然对应物 （分别为 70.5 ± 4.9% 和 76.3 ± 2.8%）。5-羟甲基胞苷 （5-hmCTP） 和 5-烯丙基胺胞苷 （5-aaCTP） 也有效抑制摄取，但效率略低于碘化胞苷 （分别为 76.3 ± 2.9% 和 64.7 ± 9.3%）。基于这些竞争性抑制数据，转运蛋白似乎相对耐受嘧啶 C5 位的取代，碘取代实际上增加了抑制。

Figure 1 图 1

Figure 1. (A) Structure of nucleobase-modified nucleoside triphosphates used for the inhibition assay; sugar and phosphates omitted for clarity. (B) Percent inhibition of uptake of [α32P]ATP (50 μM) by the analogues indicated (500 μM each). Data shown are the average and SEM of three independent trials.
图 1.（A） 用于抑制测定的核碱基修饰的核苷三磷酸的结构;为清楚起见，省略了糖和磷酸盐。（B） 所示类似物（每个 500 μM）对 [α32P] ATP （50 μM） 摄取的抑制百分比。显示的数据是三项独立试验的平均值和 SEM。

High Resolution Image
Download MS PowerPoint Slide
To explore more dramatic nucleobase modifications, we examined inhibition with thieno-CTP (thCTP) and thieno-GTP (thGTP) (Figure 1). Interestingly, both thGTP and thCTP analogues mediated relatively potent inhibition of [α32P]ATP uptake (75 ± 1.2% and 79.8 ± 1.8%, respectively). We next explored the effect of even more drastic nucleobase modifications with the unnatural triphosphates dNaMTP, d5SICSTP, and dTPT3TP, which were used to expand the genetic alphabet and code of E. coli, (24, 28, 29) as well as several precursors explored during their development (34-38) (Figure 1). The monocyclic derivatives dBENTP, d3FBTP, and dDM5TP showed little to no inhibition (≤10%), while dNaMTP and d5SICSTP showed moderate inhibition (36.6 ± 10.0%, 39.3 ± 14.0%) and dTPT3TP showed significant inhibition (72.1 ± 0.5%). Overall, these differences suggest that competitive binding is favored by large aromatic surface area, and possibly the nature of the substituent ortho to the glycosidic linkage, for example, its ability to participate in hydrogen bond (H-bond) formation, as is known to be important for polymerase recognition. (39-41) Finally, we characterized the ribonucleoside triphosphate variants, NaMTP, 5SICSTP, and TPT3TP, (29, 42) and found that 5SICSTP appeared to inhibit uptake slightly more than its deoxy counterpart (57.6 ± 10.2%), but that NaMTP and TPT3TP did not (39.8 ± 7.1% and 78.3 ± 1.5%, respectively), suggesting that the role of the 2′–OH group may be context dependent.
为了探索更显着的核碱基修饰，我们检查了噻吩-CTP （thCTP） 和噻吩-GTP （thGTP） 的抑制作用（图 1）。有趣的是，thGTP 和 thCTP 类似物都介导了对 [α32P] ATP 摄取的相对有效的抑制 （分别为 75 ± 1.2% 和 79.8 ± 1.8%）。接下来，我们探索了非天然三磷酸盐 dNaMTP、d5SICSTP 和 dTPT3TP 进行更剧烈的核碱基修饰的影响，这些三磷酸盐被用来扩展大肠杆菌的遗传字母和密码 （24， 28， 29） 以及在其发育过程中探索的几种前体 （34-38） （图 1）。单环衍生物 dBENTP 、 d3FBTP 和 dDM5TP 几乎没有抑制作用 （≤10%），而 dNaMTP 和 d5SICSTP 表现出中度抑制 （36.6 ± 10.0%、39.3 ± 14.0%）和 dTPT3TP 表现出显著抑制 （72.1 ± 0.5%）。总体而言，这些差异表明，大芳香族表面积以及可能与糖苷键邻位的取代基的性质有利于竞争性结合，例如，其参与氢键（H-键）形成的能力，众所周知，这对聚合酶识别很重要。（39-41） 最后，我们表征了核糖核苷三磷酸变体 NaMTP、5SICSTP 和 TPT3TP，（29,42），发现 5SICSTP 似乎比其脱氧对应物 （57.6 ± 10.2%） 抑制摄取略多，但 NaMTP 和 TPT3TP 没有 （39.8 ± 7.1% 和 78.3 ± 1。5%），这表明 2'-OH 基团的作用可能取决于上下文。
To further characterize the SARs associated with the sugar moiety, we explored the contribution of each hydroxyl group within the adenosine triphosphate scaffold (Figure 2). As reported previously, ATP is a more potent inhibitor of PtNTT2 than dATP (57.2 ± 7.1% vs 43.5 ± 6.6% inhibition). While ddATP showed only modest inhibition (32.2 ± 4.4%), 3′-dATP (cordyceptin) inhibits [α32P]ATP more potently than either natural analogue (71.3 ± 4.8% inhibition). This demonstrates that both OH groups contribute to competitive inhibition, but their contributions are not additive, with the 3′-OH actually reducing binding when a 2′-OH is already present.
为了进一步表征与糖部分相关的 SAR，我们探讨了三磷酸腺苷支架内每个羟基的贡献（图 2）。如前所述，ATP 是比 dATP 更有效的 PtNTT2 抑制剂 （抑制率为 57.2 ± 7.1% vs 43.5 ± 6.6%）。虽然 ddATP 仅显示适度的抑制作用 （32.2 ± 4.4%），但 3′-dATP （虫草ceptin） 比任何一种天然类似物 （71.3 ± 4.8%） 更有效地抑制 [α32P] ATP。这表明两个 OH 基团都有助于竞争性抑制，但它们的贡献不是相加的，当 2'-OH 已经存在时，3'-OH 实际上会减少结合。

Figure 2 图 2

Figure 2. (A) Structure of sugar modified nucleoside triphosphate analogues used for the inhibition assay. Base, one of the natural nucleobases; PPPO, triphosphate. (B) Percent inhibition of uptake of [α32P]ATP (50 μM) by the analogues indicated (500 μM each). Data shown are the average and SEM of three independent trials.
图 2.（A） 用于抑制测定的糖修饰核苷三磷酸类似物的结构。碱基，天然核碱基之一;PPPO，三磷酸盐。（B） 所示类似物对 [α32P] ATP （50 μM） 摄取的抑制百分比（每个 500 μM）。显示的数据是三项独立试验的平均值和 SEM。

High Resolution Image
Download MS PowerPoint Slide
To further explore the role of the 2′-substituent, we characterized analogues with altered stereochemistry or with unnatural substituents (Figure 2). We first characterized all four arabinose (Ara) triphosphates. In the case of the Ara-CTP, the altered stereochemistry increased inhibition (81.3 ± 5.2% versus 70.5 ± 4.9%), while for Ara-UTP, Ara-ATP, and Ara-GTP, it decreased inhibition (41.7 ± 15.2% versus 52.7 ± 4.4%, 33.8 ± 17.2% versus 57.2 ± 7.1%, and 47.8 ± 7.9% versus 60.4 ± 1.4%, respectively). In general, the 2′-methoxy and 2′-amino modified analogues are less potent inhibitors than their natural counterparts, with a reduction in inhibition of at least 20% in all cases relative to the parent ribonucleotide. In contrast, the 2′-fluoro (F) and 2′-azido (N3) modifications reduced binding only with the adenosine analogue (40.1 ± 5.6% and 33.4 ± 13.0% inhibition, respectively), and with the 2′-F analogue, this reduction was only to the level observed with dATP.
为了进一步探索 2′-取代基的作用，我们表征了具有改变的立体化学或不自然取代基的类似物（图 2）。我们首先表征了所有四种阿拉伯糖 （Ara） 三磷酸盐。在 Ara-CTP 的情况下，改变的立体化学增加了抑制作用（81.3 ± 5.2% 对 70.5 ± 4.9%），而对于 Ara-UTP、Ara-ATP 和 Ara-GTP，它降低了抑制作用（41.7 ± 15.2% 对 52.7 ± 4.4%、33.8 ± 17.2% 对 57.2 ± 7.1% 和 47.8 ± 7.9% 对 60.4 ± 1.4%））。一般来说，2'-甲氧基和 2'-氨基修饰的类似物是比天然对应物更有效的抑制剂，在所有情况下，相对于母体核糖核苷酸，抑制作用至少降低 20%。相比之下，2'-氟 （F） 和 2'-叠氮基 （N3） 修饰仅降低与腺苷类似物的结合（分别为 40.1 ± 5.6% 和 33.4 ± 13.0% 抑制），而使用 2'-F 类似物时，这种降低仅达到 dATP 观察到的水平。
To explore the effects of 3′-modification, we synthesized the triphosphate of azidothymidine (AZTTP, Supporting Information), and found that the 3′-N3 substituent increased inhibition relative to dTTP (56.9 ± 12.0% vs 32.9 ± 6.5%). This is in contrast to the effects at the 2′-position discussed above, where the azide substituent did not significantly alter competitive inhibition relative to dTTP.
为了探索 3'-修饰的效果，我们合成了叠氮胸苷的三磷酸盐 （AZTTP， 支持信息），发现 3'-N3 取代基相对于 dTTP 增加了抑制作用 （56.9 ± 12.0% vs 32.9 ± 6.5%）。这与上面讨论的 2′ 位的影响相反，其中叠氮化物取代基相对于 dTTP 没有显着改变竞争性抑制。
To explore more dramatic changes to the sugar, we examined the threose nucleic acid (TNA) analogues (43, 44) of the natural triphosphates (tNTPs; Figure 2), which have generated significant synthetic biology interest. (45, 46) Despite their significantly different topology, tGTP, tATP, and tTTP still competitively inhibit uptake, although significantly less so than their natural counterparts, while uptake inhibition by tCTP and CTP was virtually identical. Finally, we synthesized the triphosphate of the GTP analogue acyclovir (ACVTP, Figure 2; Supporting Information), which lacks C2′ and C3′ atoms altogether, and surprisingly, found that it inhibits ATP uptake (68.6 ± 5.5%) better than either dGTP (55.3 ± 3.6%) or GTP (60.4 ± 1.4%). This data further emphasizes the complex role of the sugar moiety.
为了探索糖的更显着变化，我们检查了天然三磷酸盐 （tNTP;图 2），这引起了合成生物学的极大兴趣。（45， 46）尽管它们的拓扑结构明显不同，但 tGTP、tATP 和 tTTP 仍然竞争性地抑制摄取，尽管明显低于它们的天然对应物，而 tCTP 和 CTP 的摄取抑制几乎相同。最后，我们合成了 GTP 类似物阿昔洛韦的三磷酸盐（ACVTP，图 2;支持信息），它完全缺乏 C2' 和 C3' 原子，令人惊讶的是，它发现它抑制 ATP 摄取 （68.6 ± 5.5%） 优于 dGTP （55.3 ± 3.6%） 或 GTP （60.4 ± 1.4%）。该数据进一步强调了糖部分的复杂作用。
Previous work by Haferkamp and co-workers demonstrated that PtNTT2 does not import ADP or AMP, (30) however its tolerance of triphosphate modification has not been explored. Thus, to examine the SARs associated with the triphosphate moiety, we characterized a variety of derivatives with intact, but modified triphosphates (Figure 3). First, we explored the ability of the α-phosphorothioate of each natural triphosphate, as well as the γ-phosphorothioate of ATP, which replaces a nonbridging oxygen atom at the α- or γ-phosphate with a sulfur atom (in each case a racemic mixture of R and S enantiomers was employed), to inhibit ATP uptake. Inhibition observed with each of these analogues was similar to that observed for the respective unmodified parent nucleotide. To explore the role of a bridging oxygen, we synthesized several analogues of AZT triphosphate in which the β,γ-bridging oxygen was replaced with a methylene (β,γ-CH2-AZT), a dichloromethylene (β,γ-CCl2-AZT), or a difluoromethylene (β,γ-CF2-AZT; Supporting Information). Such modifications have been used as biochemical probes, (47-50) as well as nonhydrolyzable triphosphate analogues. (51) In contrast to modification of a nonbridging oxygen, β,γ-CH2-AZT and β,γ-CF2-AZT showed a reduction in the inhibition of [α32P]ATP uptake relative to AZTTP (31.0 ± 20.0% and 32.6 ± 18.0%, respectively), while β,γ-CCl2-AZT showed only a slight reduction in inhibition.(50.5 ± 14.3%) Thus, though apparently context dependent, the β,γ oxygen appears to contribute more to transporter binding than the α phosphate.
Haferkamp 及其同事以前的工作表明，PtNTT2 不输入 ADP 或 AMP，（30） 但尚未探索其对三磷酸盐修饰的耐受性。因此，为了检查与三磷酸盐部分相关的 SAR，我们表征了具有完整但修饰的三磷酸盐的各种衍生物（图 3）。首先，我们探讨了每种天然三磷酸盐的 α-硫代磷酸酯以及 ATP 的 γ-硫代磷酸酯的能力，它用硫原子取代了 α-或 γ-磷酸盐处的非桥接氧原子（在每种情况下都使用了 R 和 S 对映异构体的外消旋混合物），以抑制 ATP 摄取。用这些类似物中的每一个观察到的抑制与观察到的相应未修饰的亲本核苷酸相似。为了探索桥接氧的作用，我们合成了几种 AZT 三磷酸类似物，其中β，γ-桥接氧被亚甲基 （β，γ-CH 2-AZT）、二氯亚甲基 （β，γ-CCl 2-AZT） 或二氟亚甲基 （β，γ-CF2-AZT;支持信息）。这种修饰已用作生化探针 （47-50） 以及不可水解的三磷酸类似物。（51） 与非桥接氧的修饰相比，β，γ-CH 2-AZT 和 β，γ-CF2-AZT 显示相对于 AZTTP 对 [α32P] ATP 摄取的抑制降低（分别为 31.0 ± 20.0% 和 32.6 ± 18.0%），而 β，γ-CCl2-AZT 仅显示抑制略有降低。（50.5 ± 14.3%）因此，尽管显然依赖于环境，但 β，γ 氧似乎比磷酸α对转运蛋白结合的贡献更大。

Figure 3 图 3

Figure 3. (A) Structure of triphosphate-modified nucleoside triphosphates used for the inhibition assay. Base, one of the natural nucleobases. (B) Percent inhibition of uptake of [α32P]ATP (50 μM) by the analogues indicated (500 μM each). Data shown are the average and SEM of three independent trials.
图 3.（A） 用于抑制测定的三磷酸修饰的核苷三磷酸的结构。碱基，天然核碱基之一。（B） 所示类似物对 [α32P] ATP （50 μM） 摄取的抑制百分比（每个 500 μM）。显示的数据是三项独立试验的平均值和 SEM。

High Resolution Image
Download MS PowerPoint Slide

Direct Analysis of Uptake
直接分析摄取

The traditional assay to directly characterize triphosphate uptake relies on radiolabeled substrates and is straightforward to employ. Similarly, the analysis of intrinsically fluorescent analogues is also straightforward. To use these assays to characterize PtNTT2 SARs, we explored the uptake of thCTP and thGTP, which are intrinsically fluorescent, and 5-IdCTP radiolabeled with 125I (Supporting Information). We found that the thCTP and thGTP are imported more efficiently than 5-IdCTP, consistent with PtNTT2 efficiently importing analogues bearing natural-like nucleobases with increased aromatic surface area, and suggesting that the potent inhibition mediated by 5-ICTP results from nonproductive binding.
直接表征三磷酸盐摄取的传统测定依赖于放射性标记的底物，并且易于使用。同样，本征荧光类似物的分析也很简单。为了使用这些测定来表征 PtNTT2 SARs，我们探索了 thCTP 和 thGTP 的摄取，它们是固有荧光的，以及用 125I 放射性标记的 5-IdCTP （支持信息）。我们发现 thCTP 和 thGTP 的导入效率高于 5-IdCTP，这与 PtNTT2 有效导入带有增加芳香族表面积的天然样核碱基的类似物一致，并表明 5-ICTP 介导的有效抑制是由非生产性结合引起的。
Many triphosphates of interest are neither intrinsically fluorescent nor readily amenable to radiolabeling. Thus, we explored the development of a quantitative LC-MS/MS assay that would permit the general characterization of modified nucleoside triphosphate uptake. Briefly, exponentially growing YZ2 cells were treated with varying concentrations of nucleoside triphosphate and incubated for 1 h at 37 °C. Cells were then pelleted and washed before extracting intracellular nucleotides with acidic acetonitrile. (52) Nucleotides were then fully degraded to their corresponding nucleosides through the addition of shrimp alkaline phosphatase, and then quantified by LC-MS/MS. To correct for any passive uptake of the free nucleoside produced by extracellular degradation of the triphosphate, we subtracted the signal from a no transporter BL21(DE3) CmR control strain for each concentration of added triphosphate. The concentration of nucleoside detected thus represents the amount of triphosphate imported, and includes all factors that may affect import, such as extracellular degradation and diffusion into the periplasm. Initial apparent velocities were determined using an external calibration curve constructed using free nucleosides and then plotted against the concentration of triphosphate added to the media. The resulting curves were fit to the Michaelis–Menten equation to determine apparent KM (μM) and apparent Vmax (fmol·cell–1·hr–1) values (Table 1), with Vmax/KM being taken as a measure of uptake efficiency.
许多感兴趣的三磷酸盐既不是固有的荧光，也不容易进行放射性标记。因此，我们探索了定量 LC-MS/MS 分析的开发，该分析将允许对修饰的三磷酸核苷摄取进行一般表征。简而言之，用不同浓度的三磷酸核苷处理指数生长的 YZ2 细胞，并在 37 °C 下孵育 1 小时。 然后对细胞进行沉淀和洗涤，然后用酸性乙腈提取细胞内核苷酸。（52） 然后通过添加虾碱性磷酸酶将核苷酸完全降解为相应的核苷，然后通过 LC-MS/MS 进行定量。为了校正三磷酸盐细胞外降解产生的游离核苷的任何被动摄取，我们从无转运蛋白 BL21（DE3） CmR 对照菌株中减去每种浓度的添加三磷酸盐的信号。因此，检测到的核苷浓度代表输入的三磷酸盐的量，并包括可能影响输入的所有因素，例如细胞外降解和扩散到周质中。使用使用游离核苷构建的外部校准曲线确定初始表观速度，然后根据添加到培养基中的三磷酸盐浓度进行绘图。将所得曲线拟合到 Michaelis-Menten 方程中，以确定表观 KM （μM） 和表观 Vmax （fmol·cell–1·hr–1） 值（表 1），其中 Vmax/KM 作为摄取效率的量度。
Table 1. Rates of Uptake for Various Nucleoside Triphosphates by PtNTT2 As Determined by LC-MS/MSa
表 1.通过 LC-MS/MSa 测定 PtNTT2 对各种核苷三磷酸的摄取速率
triphosphate 三磷酸盐Vmax (fmol·cell–1·hr–1)
V max （fmol·cell–1·hr–1）		KM (μM)
KM （微米）		Vmax/KM
ATPb
ATP乙		(1.74 ± 0.091) × 10–2
（1.74 ± 0.091） × 10–2		186.0 ± 21.1(9.34 ± 0.32) × 10–5
（9.34 ± 0.32） × 10–5
dATP(1.24 ± 0.34) × 10–3
（1.24 ± 0.34） × 10–3		365.8 ± 47.9(3.38 ± 0.54) × 10–6
（3.38 ± 0.54） × 10-6
3′-dATP(1.04 ± 0.29) × 10–3
（1.04 ± 0.29） × 10–3		162.7 ± 58.8(6.41 ± 0.31) × 10–6
（6.41 ± 0.31） × 10-6
ddATP ddATP 抗体(2.3 ± 0.81) × 10–3
（2.3 ± 0.81） × 10–3		766.6 ± 374.7(3.00 ± 0.74) × 10–6
（3.00 ± 0.74） × 10–6
2′-F ATP(7.75 ± 2.3) × 10–5
（7.75 ± 2.3） × 10-5		478.7 ± 262.4(1.64 ± 0.28) × 10–7
（1.64 ± 0.28） × 10–7
tATP–c–c(1.21 ± 0.17) × 10–7
（1.21 ± 0.17） × 10–7
c3dATP(1.75 ± 0.21) × 10–4
（1.75 ± 0.21） × 10–4		388.2 ± 74.6(4.81 ± 0.96) × 10–7
（4.81 ± 0.96） × 10–7
c7dATP–c–c(8.75 ± 1.3) × 10–7
（8.75 ± 1.3） × 10-7
dNaM d南–c–c(1.94 ± 0.15) × 10–8
（1.94 ± 0.15） × 10–8
dTPT3(7.50 ± 0.73) × 10–5
（7.50 ± 0.73） × 10–5		116.9 ± 11.4(6.68 ± 1.38) × 10–7
（6.68 ± 1.38） × 10–7
a

Data shown are the average and SEM of three independent trials.


a

显示的数据是三项独立试验的平均值和 SEM。

b

Assayed by radiolabeling.


b

通过放射性标记进行测定。

c

Not determined.


c

未确定。

To validate the assay, we first characterized the uptake of dATP using [α32P]dATP and the standard radioactivity-based assay (Supporting Information). Uptake of dATP was found to proceed with a KM of 171 ± 78.4 μM and a Vmax of 1.88 ± 0.49 × 10–3 fmol·cell–1·hr–1. This KM is in good agreement with that reported previously (270.6 μM). (30) With the LC-MS/MS assay, we found the uptake of dATP to proceed with a KM of 366 ± 48 μM and a Vmax of 1.24 ± 0.34 × 10–3 fmol·cell–1·hr–1, which we deemed to be in sufficient agreement with the results of the conventional assay. Using the validated LC-MS/MS assay, we proceeded to characterize the uptake of 3′-dATP, which we found to proceed with a Vmax that is virtually identical to that of dATP (1.04 ± 0.29 × 10–3 fmol·cell–1·hr–1) and a KM that is 2-fold reduced (163 ± 58.8 μM). We next characterized the uptake of ddATP, which we found to proceed with a 2-fold increase in both Vmax and KM (2.3 ± 0.81 × 10–3 fmol·cell–1·hr–1 and 767 ± 375 μM, respectively), relative to dATP. Comparison of ATP, dATP, 3′-dATP, and ddATP reveals that while both hydroxyl groups contribute similarly to turnover, the 2′-OH contributes more to productive binding, with the 3′-OH contributing only if the 2′-OH is absent. The data also suggests that the uniquely potent inhibition of transporter function mediated by 3′-dATP (see above) results from nonproductive binding, and that the 3′-OH plays an important role by reducing it. Regardless, both ddATP and 3′-dATP are imported with second order rate constants that are similar to or even greater than that for the import of dATP.
为了验证该测定，我们首先使用 [α32P]dATP 和基于放射性的标准测定（支持信息）表征了 dATP 的摄取。发现 dATP 的摄取在 171 ± 78.4 μM 的 KM 和 1.88 ± 0.49 的 Vmax × 10-3 fmol·cell–1·hr–1 进行。该 KM 与之前报道的 （270.6 μM） 非常一致。（30） 通过 LC-MS/MS 分析，我们发现 dATP 的摄取在 10-3 fmol·cell–1·hr–1 × 366 ± 48 μM 的 K M 和 1.24 ± 0.34 的 V max 进行，我们认为这与常规测定的结果充分一致。使用经过验证的 LC-MS/MS 分析，我们继续表征 3′-dATP 的摄取，我们发现其 Vmax 与 dATP 几乎相同（1.04 ± 0.29 × 10-3 fmol·cell–1·hr–1）和 KM 减少 2 倍（163 ± 58.8 μM）。接下来，我们表征了 ddATP 的摄取，我们发现相对于 dATP，ddATP 的 Vmax 和 KM 都增加了 2 倍（分别为 2.3 ± 0.81 × 10-3 fmol·cell–1·hr–1 和 767 ± 375 μM）。ATP、dATP、3′-dATP 和 ddATP 的比较表明，虽然两个羟基对转换的贡献相似，但 2′-OH 对生产性结合的贡献更大，而 3′-OH 仅在不存在 2′-OH 时发挥作用。 数据还表明，由 3′-dATP（见上文）介导的对转运蛋白功能的独特有效抑制是由非生产性结合引起的，并且 3′-OH 通过减少它发挥重要作用。无论如何，ddATP 和 3′-dATP 导入时的二阶速率常数与 dATP 导入的二阶速率常数相似甚至更大。
To further explore the role of the 2′-substituent, we examined the uptake of the 2′-F analogue of adenosine triphosphate. Import of 2′-F ATP proceeded with a KM that is similar to that observed for dATP (478 ± 262 μM), but with a Vmax that is 16-fold reduced (7.75 ± 2.3 × 10–5 fmol·cell–1·hr–1). Thus, while the addition of a 2′-OH to dATP (i.e., ATP) slightly increases binding (2-fold) and significantly increases turnover (14-fold), a fluoro substituent at the same position has little effect on binding but a significant and opposite effect on turnover. The deleterious effect on turnover is unlikely to result from an alteration of sugar conformation (both substituents favor the C3′-endo conformation of the sugar ring (53)) and thus must result from differences in size, electronegativity, and perhaps most likely, H-bonding potential. Regardless, 2′-F ATP is imported with a second order rate constant that is only ∼20-fold reduced relative to dATP.
为了进一步探索 2'-取代基的作用，我们检查了三磷酸腺苷的 2'-F 类似物的摄取。2′-F ATP 的导入与 dATP 观察到的 KM 相似 （478 ± 262 μM），但 Vmax 降低了 16 倍（7.75 ± 2.3 × 10–5 fmol·cell–1·hr–1）。因此，虽然向 dATP（即 ATP）中添加 2′-OH 略微增加结合（2 倍）并显着增加周转率（14 倍），但相同位置的含氟取代基对结合几乎没有影响，但对周转有显着且相反的影响。对周转的有害影响不太可能是由糖构象的改变引起的（两个取代基都有利于糖环的 C3′-内构象 （53）），因此必须由大小、电负性以及最有可能的 H 键电位的差异引起。无论如何，2′-F ATP 是以二阶速率常数导入的，相对于 dATP 仅减少了 ∼20 倍。
As a final examination of sugar modifications, we explored the uptake of the TNA analogue tATP, which we found was too inefficient to determine KM and a Vmax independently. However, we were able to determine the second order rate constant, which was found to be 1.21 ± 0.17 × 10–7 mL·cell–1·hr–1, which is only ∼30-fold reduced relative to dATP.
作为糖修饰的最终检查，我们探索了 TNA 类似物 tATP 的摄取，我们发现它的效率太低，无法独立确定 KM 和 Vmax。然而，我们能够确定二阶速率常数，发现它是 1.21 ± 0.17 × 10-7 mL·cell–1·hr-1，相对于 dATP 仅减少了 ∼30 倍。
To explore the effects of nucleobase modifications, we characterized the uptake of c3dATP and c7dATP. The 3-deaza analogue was imported with a KM of 388 ± 75 μM and a Vmax of 1.75 ± 0.21 × 10–4 fmol·cell–1·hr–1, while the 7-deaza analogue was imported with a KM too high to determine (>1 mM), but with a Vmax/KM of 8.75 ± 1.3 × 10–7 mL·cell–1·hr–1. Thus, the elimination of N7 results in an at least 3-fold reduction in binding with little effect on turnover, while elimination of N3 has little effect on binding, but reduces turnover 7-fold. This suggests that the increased competitive inhibition observed with the nucleotides with bicyclic unnatural nucleobase analogues, relative to monocyclic analogues (see above), likely results from increased aromatic surface area. Regardless, these analogues are imported with an efficiency that is only 4–7-fold reduced relative to dATP.
为了探索核碱基修饰的影响，我们表征了 c3dATP 和 c7dATP 的摄取。导入 3-deaza 类似物，KM 为 388 ± 75 μM，V max 为 1.75 ± 0.21 × 10–4 fmol·cell–1·hr–1，而 7-deaza 类似物导入时，KM 太高而无法确定 （>1 mM），但 Vmax/KM 为 8.75 ± 1.3 × 10–7 mL·cell–1·hr–1.因此，消除 N7 导致结合减少至少 3 倍，对周转影响很小，而消除 N3 对结合影响不大，但将周转减少 7 倍。这表明，相对于单环类似物（见上文），用双环非天然核碱基类似物的核苷酸观察到的竞争性抑制增加可能是由于芳香族表面积增加所致。无论如何，这些类似物的进口效率相对于 dATP 仅降低 4-7 倍。
Finally, we demonstrated previously that the PtNTT2-mediated import of dNaMTP, d5SICSTP, and dTPT3TP is sufficient to enable replication of DNA containing the corresponding unnatural base pair. (24, 28, 29) In addition, the inhibition data presented above demonstrates that PtNTT2 effectively binds these unnatural triphosphates, as well as others with predominantly hydrophobic nucleobases that are very different from natural nucleobases. Using the LC-MS/MS assay, we observed a KM for dNaMTP that was too high to determine independently of Vmax (KM > 1 mM), but the Vmax/KM was found to be 1.94 ± 0.15 × 10–8 mL·cell–1·hr–1. In contrast, dTPT3TP was found to be imported more efficiently, with a KM of 117 ± 11.4 μM and a Vmax of 7.50 ± 0.73 × 10–5 fmol·cell–1·hr–1. The more efficient import of dTPT3TP may result from the altered size, electrostatics, or H-bonding potential of its nucleobase.
最后，我们之前证明，PtNTT2 介导的 dNaMTP、d5SICSTP 和 dTPT3TP 的输入足以复制含有相应非天然碱基对的 DNA。（24、28、29）此外，上述抑制数据表明，PtNTT2 可有效结合这些非天然三磷酸盐，以及与天然核碱基截然不同的其他主要疏水核碱基的三磷酸盐。使用 LC-MS/MS 分析，我们观察到 dNaMTP 的 KM 太高，无法单独确定 Vmax （KM > 1 mM），但发现 Vmax/KM 为 1.94 ± 0.15 × 10–8 mL·cell–1·hr–1。相比之下，发现 dTPT3 TP 的导入效率更高，在 10-5 fmol·cell–1·hr–1 ×，K M 为 117 ± 11.4 μM，V max 为 7.50 ± 0.73。dTPT3TP 的更高效导入可能是由于其核碱基的尺寸、静电或 H 键电位的改变所致。

Use of PtNTT2 to Study Replication and Transcription in Vivo
使用 PtNTT2 研究体内复制和转录

The specificity of DNA and RNA polymerases is ubiquitously studied in vitro with model polymerases that are amenable to recombinant expression. However, this does not include the main replicative polymerases, which thus have not been extensively characterized. Moreover, it is not clear whether behavior in vitro translates to the natural in vivo environment. Thus, to explore replication in vivo, studies typically rely on the uptake of modified nucleotides and their subsequent three-step activation to the corresponding triphosphates. To explore the more controlled delivery of triphosphates, E. coli YZ2 harboring a pUC19 plasmid was grown to an OD600 of ∼0.3 in the presence of an individual [α32P]-labeled dNTP or NTP, and the bulk RNA or plasmid DNA was then recovered and analyzed by PAGE and phosphorimaging to quantify the extent of radiolabeling.
DNA 和 RNA 聚合酶的特异性在体外普遍使用适合重组表达的模型聚合酶进行研究。然而，这不包括主要的复制聚合酶，因此尚未对其进行广泛表征。此外，目前尚不清楚体外行为是否转化为自然体内环境。因此，为了探索体内复制，研究通常依赖于修饰核苷酸的摄取及其随后对相应三磷酸盐的三步激活。为了探索三磷酸盐的更受控递送，在个体 [α32P] 标记的 dNTP 或 NTP 存在下，将含有 pUC19 质粒的大肠杆菌 YZ2 生长至 ∼0.3 的 OD600，然后回收大量 RNA 或质粒 DNA 并通过 PAGE 和磷光成像分析以量化放射性标记的程度。
Analysis of bulk RNA revealed that the addition of each [α32P]-labeled NTP resulted in efficient RNA labeling (Figure 4). Interestingly, we also observed significant labeling of plasmid DNA with each NTP. The addition of [α32P]-labeled dNTPs resulted in low but detectable levels of RNA labeling, and as expected, the import of [α32P]dGTP, [α32P]dATP, and [α32P]dTTP resulted in the efficient labeling of plasmid DNA (Figure 4). Interestingly, through comparison of the extent of labeling, it was clear that with the exception of [α32P]GTP, the addition of each [α32P]dNTP resulted in the less efficient labeling of DNA than the corresponding NTP. One possible explanation for this cross-compartment labeling is greater import and/or degradation and subsequent phosphate exchange with the NTP substrates.
大量 RNA 的分析表明，添加每个 [α32P] 标记的 NTP 可实现高效的 RNA 标记（图 4）。有趣的是，我们还观察到每个 NTP 对质粒 DNA 的显著标记。添加 [α32P] 标记的 dNTP 导致 RNA 标记水平较低但可检测，正如预期的那样，[α32P]dGTP、[α32P]dATP 和 [α32P]dTTP 的导入导致了质粒 DNA 的高效标记（图 4）。有趣的是，通过比较标记程度，很明显，除了 [α32P]GTP 外，添加每个 [α32P]dNTP 会导致 DNA 标记的效率低于相应的 NTP。这种跨区室标记的一种可能解释是更大的输入和/或降解以及随后与 NTP 底物的磷酸盐交换。

Figure 4 图 4

Figure 4. RNA and DNA radiolabeled with [α32P]-labeled analogues of each natural nucleotide.
图 4.用每种天然核苷酸的 [α32P] 标记的类似物放射性标记的 RNA 和 DNA。

High Resolution Image
Download MS PowerPoint Slide
The results with the addition of [α32P]dCTP were strikingly different than with the other [α32P]-labeled dNTPs. In this case we observed significant toxicity (Figure S5), and even after normalization for the amount of DNA analyzed, we observed significantly less radioactivity in the recovered DNA (Figure 4). Previous studies have demonstrated PtNTT2 imports dCTP more efficiently than the other dNTPs (30), indicating that these observations are not related to low levels of uptake. To more directly explore the fate of the imported dCTP, we examined the incorporation of dC into RNA using an adaptation of the developed LC-MS/MS assay. Briefly, E. coli YZ2 was grown in 2×YT media, and at an OD600 of 0.3, 250 μM of dCTP was added. After 90 min of growth, cellular RNA was extracted and degraded using a mixture of benzonase, phosphodiesterase I, and calf intestinal phosphatase, a cocktail which has been shown to nonspecifically degrade even modified nucleic acids to free nucleosides. (54, 55) The degraded nucleosides were then analyzed by LC-MS/MS to determine the level of dC incorporation relative to C (Table 2). Interestingly, we found that 3% of the C had been replaced with dC. This clearly indicates that at the elevated concentrations of dCTP afforded by PtNTT2, dCTP is used by E. coli RNA polymerase only ∼30-fold less efficiently than the corresponding natural substrate, and also demonstrates that at least some of the cross-compartment labeling observed with the radioactive substrates results from actual nucleotide incorporation, as opposed to phosphate exchange. This makes the above-described inefficient labeling of DNA with [α32P]dCTP all the more surprising, and it suggests that the labeling of RNA may be more efficient than the labeling of DNA, possibly due to differences in the polymerases and/or their accessibility
添加 [α32P]dCTP 的结果与其他 [α32P] 标记的 dNTP 的结果截然不同。在这种情况下，我们观察到明显的毒性（图 S5），即使在分析的 DNA 量标准化后，我们观察到回收的 DNA 中的放射性明显降低（图 4）。先前的研究表明，PtNTT2 比其他 dNTP 更有效地输入 dCTP （30），这表明这些观察结果与低水平摄取无关。为了更直接地探索进口 dCTP 的命运，我们使用开发的 LC-MS/MS 分析的改编版检查了 dC 掺入 RNA 的过程。简而言之，大肠杆菌 YZ2 在 2×YT 培养基中生长，OD 600 为 0.3，加入 250 μM dCTP。生长 90 分钟后，使用苯并酶、磷酸二酯酶 I 和小牛肠磷酸酶的混合物提取和降解细胞 RNA，这种混合物已被证明可以非特异性地将修饰的核酸降解为游离核苷。（54， 55）然后通过 LC-MS/MS 分析降解的核苷，以确定相对于 C 的 dC 掺入水平（表 2）。有趣的是，我们发现 3% 的 C 已被 dC 取代。这清楚地表明，在 PtNTT2 提供的高浓度 dCTP 下，大肠杆菌 RNA 聚合酶对 dCTP 的利用效率仅比相应的天然底物低 ∼30 倍，并且还表明至少一些用放射性底物观察到的跨区室标记是由实际核苷酸掺入引起的，而不是磷酸盐交换。这使得上述用 [α32P]dCTP 对 DNA 进行低效标记更加令人惊讶，并且它表明 RNA 的标记可能比 DNA 的标记更有效，这可能是由于聚合酶和/或其可及性的差异
Table 2. Percent Labeling of RNA and DNA from Nucleoside Triphosphates Imported by PtNTT2 Relative to the Corresponding Natural Nucleotidea
表 2.PtNTT2 导入的三磷酸核苷的 RNA 和 DNA 相对于相应天然核苷酸a 的标记百分比
triphosphate 三磷酸盐RNA labeling (%) RNA 标记 （%）DNA labeling (%) DNA 标记 （%）
2′-F UTP 2′-F 双绞线<0.3–b
2′-F GTP1.58 ± 0.16–b
2′-F ATP0.78 ± 0.19–b
2′-F CTP9.11 ± 0.982.77 ± 0.16
dCTP3.03 ± 0.29–b
a

Data shown are the average and SEM of three independent trials.


a

显示的数据是三项独立试验的平均值和 SEM。

b

Not determined.


b

未确定。

Based on the growing interest in the replacement of the natural nucleotides of DNA or RNA in an organism with modified analogues, (54-58) we explored the import and fate of 2′-F modified triphosphates. We first examined the import and incorporation of each unnatural 2′-F triphosphate into RNA, using the same assay described above, but with the addition of 250 μM of 2′-F ATP, 2′-F GTP, 2′-F UTP, or 2′-F CTP to the media. While the addition of the 2′-F ATP, GTP, or UTP analogues had little effect on growth, the addition of the 2′-F CTP analogue resulted in a reduced rate of growth (Figure S5). Upon analysis, we were unable to detect the incorporation of 2′-F U, but 2′-F A and 2′-F G were detected at 0.8% and 1.6% of the level of their natural counterparts, respectively, and remarkably, 2′-F C was detected at greater than 9%. (Table 2) As with its radiolabeled counterpart, the toxicity and greater incorporation of the analogue likely results from increased import by PtNTT2. Indeed, cell growth and analogue incorporation levels similar to those observed with the other modified NTPs were observed with the addition of less 2′-F CTP to the media (Supporting Information). This data demonstrates that the 2′-F modified substrates, at their imported concentrations, are incorporated into RNA only ∼10 to 100-fold less efficiently than the natural NTP substrates.
基于人们对用修饰的类似物替换生物体中 DNA 或 RNA 的天然核苷酸的日益增长的兴趣，（54-58） 我们探索了 2′-F 修饰三磷酸的输入和命运。我们首先使用上述相同的测定法检查了每种非天然 2'-F 三磷酸盐的导入和掺入 RNA，但向培养基中添加了 250 μM 的 2'-F ATP、2'-F GTP、2'-F UTP 或 2'-F CTP。虽然添加 2'-F ATP、GTP 或 UTP 类似物对生长影响不大，但添加 2'-F CTP 类似物导致生长速率降低（图 S5）。经过分析，我们无法检测到 2'-F U 的掺入，但检测到 2'-F A 和 2'-F G 的水平分别为天然对应物水平的 0.8% 和 1.6%，值得注意的是，检测到 2'-F C 的水平大于 9%。（表 2）与其放射性标记的对应物一样，类似物的毒性和更大的掺入可能是由于 PtNTT2 的输入增加造成的。事实上，在培养基中添加较少的 2′-F CTP 时，观察到细胞生长和类似物掺入水平与其他修饰的 NTP 相似（支持信息）。该数据表明，在输入浓度下，2′-F 修饰底物掺入 RNA 的效率仅比天然 NTP 底物低 ∼10 至 100 倍。
The data described above suggest that providing modified substrates for DNA synthesis may be more challenging than for RNA synthesis. Thus, to determine whether a 2′-F substrate could be incorporated into DNA, we grew E. coli YZ2 harboring a pUC19 plasmid in media containing 250 μM 2′-F CTP. Proceeding as above, we found that 2.77 ± 0.16% of the dC residues of pUC19 were replaced with 2′-F C (Table 2). Thus, along with our previously reported results with dNaMTP, d5SICSTP, and dTPT3TP, (24, 28, 29) this data demonstrates that PtNTT2 imported substrates may access an active replication fork and participate in the synthesis of significant amounts of modified DNA, although apparently less efficiently than with RNA.
上述数据表明，为 DNA 合成提供修饰底物可能比 RNA 合成更具挑战性。因此，为了确定 2′-F 底物是否可以掺入 DNA 中，我们在含有 250 μM 2′-F CTP 的培养基中培养了含有 pUC19 质粒的大肠杆菌 YZ2。如上所述，我们发现 pUC19 的 2.77 ± 0.16% 的 dC 残基被 2'-F C 取代（表 2）。因此，连同我们之前报道的 dNaMTP、d5SICSTP 和 dTPT3TP 的结果 （24， 28， 29），该数据表明 PtNTT2 输入的底物可以访问活性复制叉并参与大量修饰 DNA 的合成，尽管效率明显低于 RNA。

Conclusions 结论

Click to copy section link
单击以复制部分链接Section link copied!
The ability to directly import nucleoside triphosphates into E. coli allows for the controlled introduction of labeled or modified nucleotides for molecular or synthetic biological applications. However, many potential applications depend on the import of modified analogues, and thus on the substrate scope of PtNTT2. The competitive binding SAR data presented here demonstrates that susbstituents at the 5-position are generally well tolerated. The data also reveals that increased nucleobase surface area can be tolerated, as can at least in some cases, dramatic alterations. At the sugar, both 2′- and 3′-OH groups contribute to competitive binding, but in a nonadditive manner, while other substituents, or even other sugar topologies, can have variable effects, depending on the type of modification and the nucleotide scaffold. Finally, the β,γ-bridging oxygen appears to be generally more important for binding than either the α- or γ-nonbridging oxygen, potentially providing an important mechanism to control the specificity of nucleoside triphosphates relative to di- and monophosphates.
将三磷酸核苷直接导入大肠杆菌的能力允许受控引入标记或修饰的核苷酸，用于分子或合成生物应用。然而，许多潜在的应用取决于改性类似物的进口，因此取决于 PtNTT2 的底物范围。此处提供的竞争性结合 SAR 数据表明，5 位的三元通常耐受性良好。数据还显示，核碱基表面积的增加是可以容忍的，至少在某些情况下可以容忍剧烈的变化。在糖中，2′-和 3′-OH 基团都有助于竞争性结合，但以非加成方式，而其他取代基，甚至其他糖拓扑结构，可能具有不同的影响，具体取决于修饰类型和核苷酸支架。最后，β，γ-桥接氧似乎通常比 α 或 γ-非桥接氧更重要，这可能提供了一种重要的机制来控制三磷酸核苷相对于二磷酸盐和一磷酸盐的特异性。
While convenient to measure, uptake inhibition is a poor proxy for import, and the direct SARs generated using the developed LC-MS/MS assay in fact reveal that the uptake inhibition assay is complicated by significant nonproductive binding. This direct SAR data is to our knowledge the first reported for a transporter, and it reveals that while the H-bond acceptor imine moieties of a nucleobase can facilitate uptake, either by increased binding or turnover, at least in the cases examined they are not required for efficient import. It also reveals that while both ribotyl hydroxyl groups facilitate uptake, the 3′-OH only contributes in the absence of a 2′-OH, and again, that neither are actually required for efficient uptake. While we were unable to characterize the role of the triphosphate moieties with the LC-MS/MS assay, as the analysis currently relies on their removal, the data clearly indicate that the majority of PtNTT2 substrate recognition is focused on the this region, which is consistent with the uptake inhibition SAR data.
虽然易于测量，但摄取抑制并不是输入的不良代表，并且使用开发的 LC-MS/MS 测定生成的直接 SAR 实际上表明，摄取抑制测定因显著的非生产性结合而复杂。据我们所知，这种直接 SAR 数据是首次报道的转运蛋白，它揭示了虽然核碱基的 H 键受体亚胺部分可以通过增加结合或周转来促进摄取，但至少在检查的情况下，它们不是有效输入所必需的。它还揭示了虽然两个核脂酰羟基都促进摄取，但 3'-OH 仅在没有 2'-OH 的情况下发挥作用，而且，两者都不是有效摄取所必需的。虽然我们无法用 LC-MS/MS 分析来表征三磷酸基团的作用，因为目前的分析依赖于它们的去除，但数据清楚地表明，大多数 PtNTT2 底物识别集中在该区域，这与摄取抑制 SAR 数据一致。
Our initial study of the utility of the NTT not only demonstrated that the imported triphosphates can be used by the cell, but also revealed that the efficiency with which NTPs label RNA relative to DNA is much higher than the efficiency with which dNTPs label DNA relative to RNA. While this may in part be due to phosphate exchange, the surprisingly efficient incorporation of dC into RNA with the import of dCTP demonstrates a significant level of polymerase-mediated nucleotide incorporation. It is thus possible that the increased efficiency with which RNA is labeled may be due to decreased substrate specificity of RNA polymerase, which is consistent with the lower fidelity of transcription versus replication. However, another more provocative explanation is that while the total cellular concentration of NTPs may be available for transcription, only a more difficult to access subpopulation of the total dNTP pool may be available for replication. Indeed, DNA replication is highly compartmentalized, and supramolecular complexes of proteins involved in dNTP synthesis and replication have been identified and are thought to produce dNTPs from their ribonucletide precursors, and then channel them directly to the replication fork. (59, 60) Nonetheless, the data demonstrates that both RNA and DNA are labeled by 2′-F CTP, suggesting that PtNTT2 may be used to study the synthesis of DNA or RNA.
我们对 NTT 效用的初步研究不仅表明输入的三磷酸盐可以被细胞使用，而且还揭示了 NTP 相对于 DNA 标记 RNA 的效率远高于 dNTP 相对于 RNA 标记 DNA 的效率。虽然这可能部分是由于磷酸盐交换，但 dC 与 dCTP 的导入令人惊讶地有效地掺入 RNA 中，这表明聚合酶介导的核苷酸掺入水平很高。因此，RNA 标记效率的提高可能是由于 RNA 聚合酶的底物特异性降低，这与转录与复制的保真度较低一致。然而，另一个更具挑衅性的解释是，虽然 NTP 的总细胞浓度可能可用于转录，但只有总 dNTP 池中更难接近的亚群可用于复制。事实上，DNA 复制是高度区室化的，并且已经确定了参与 dNTP 合成和复制的蛋白质的超分子复合物，并且被认为可以从其核糖核细胞前体产生 dNTP，然后将它们直接引导到复制叉。（59， 60）尽管如此，数据表明 RNA 和 DNA 都由 2′-F CTP 标记，表明 PtNTT2 可用于研究 DNA 或 RNA 的合成。
Perhaps most importantly, the data clearly demonstrate that modified nucleoside triphosphates can be made available within E. coli. In fact, of all the analogues examined, which include analogues with a wide range of nucleobase and sugar modifications, the efficiency of dNaMTP import is the lowest; however, we have already shown that the import of dNaMTP is sufficient to support the replication of an unnatural base pair on a high copy plasmid. Thus, the data suggest that all of the analogues tested, and likely many others, are likely to be imported into E. coli by PtNTT2 with an efficiency that is sufficient for molecular and synthetic biology applications.
也许最重要的是，数据清楚地表明，修饰的核苷三磷酸可以在大肠杆菌中提供。事实上，在所有检查的类似物中，包括具有广泛核碱基和糖修饰的类似物，dNaMTP 导入的效率是最低的;然而，我们已经表明，dNaMTP 的导入足以支持在高拷贝质粒上复制非天然碱基对。因此，数据表明，所有测试的类似物，可能还有许多其他类似物，都可能通过 PtNTT2 输入到大肠杆菌中，其效率足以满足分子和合成生物学应用的需求。

Supporting Information 支持信息

Click to copy section link
单击以复制部分链接Section link copied!

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b11404.
支持信息可在 ACS Publications 网站上免费获取，网址为 DOI：10.1021/jacs.7b11404。

  • Scheme S1, Methods, Supporting Figures S1–S6, Supporting Tables S1 and S2, Supporting References (PDF)
    方案 S1，方法，支持图 S1-S6，支持表 S1 和 S2，支持参考文献 （PDF）

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

Click to copy section linkSection link copied!
  • Corresponding Author
  • Authors
    • Aaron W. Feldman - Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States;  Orcidhttp://orcid.org/0000-0002-9495-2357
    • Emil C. Fischer - Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
    • Michael P. Ledbetter - Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
    • Jen-Yu Liao - Department of Pharmaceutical Sciences, University of California, Irvine, California 92697, United States
    • John C. Chaput - Department of Pharmaceutical Sciences, University of California, Irvine, California 92697, United States;  Orcidhttp://orcid.org/0000-0003-1393-135X
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

Click to copy section linkSection link copied!

Financial support was provided by the National Institutes of Health (GM118178 to F.E.R.) and the National Science Foundation Graduate Research Fellowship Program (DGE-1346837 to A.W.F. and M.P.L.)

References

Click to copy section linkSection link copied!

This article references 60 other publications.

  1. 1
    Chen, T.; Hongdilokkul, N.; Liu, Z.; Thirunavukarasu, D.; Romesberg, F. E. Curr. Opin. Chem. Biol. 2016, 34, 80– 87 DOI: 10.1016/j.cbpa.2016.08.001
    Google Scholar
    1
    The expanding world of DNA and RNA
    Chen, Tingjian; Hongdilokkul, Narupat; Liu, Zhixia; Thirunavukarasu, Deepak; Romesberg, Floyd E.
    Current Opinion in Chemical Biology (2016), 34 (), 80-87CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    DNA and RNA are remarkable because they can both encode information and possess desired properties, including the ability to bind specific targets or catalyze specific reactions. Nucleotide modifications that do not interfere with enzymic synthesis are now being used to bestow DNA or RNA with properties that further increase their utility, including phosphate and sugar modifications that increase nuclease resistance, nucleobase modifications that increase the range of activities possible, and even whole nucleobase replacement that results in selective pairing and the creation of unnatural base pairs that increase the information content. These modifications are increasingly being applied both in vitro and in vivo, including in efforts to create semi-synthetic organisms with altered or expanded genetic alphabets.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVWmtr%252FO&md5=442970017f6d14ab44454a16bcb86303
  2. 2
    Houlihan, G.; Arangundy-Franklin, S.; Holliger, P. Acc. Chem. Res. 2017, 50, 1079– 1087 DOI: 10.1021/acs.accounts.7b00056
    Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Malyshev, D. A.; Romesberg, F. E. Angew. Chem., Int. Ed. 2015, 54, 11930– 11944 DOI: 10.1002/anie.201502890
    Google Scholar
    3
    The expanded genetic alphabet
    Malyshev, Denis A.; Romesberg, Floyd E.
    Angewandte Chemie, International Edition (2015), 54 (41), 11930-11944CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. All biol. information, since the last common ancestor of all life on Earth, has been encoded by a genetic alphabet consisting of only four nucleotides that form two base pairs. Long-standing efforts to develop two synthetic nucleotides that form a third, unnatural base pair (UBP) have recently yielded three promising candidates, one based on alternative hydrogen bonding, and two based on hydrophobic and packing forces. All three of these UBPs are replicated and transcribed with remarkable efficiency and fidelity, and the latter two thus demonstrate that hydrogen bonding is not unique in its ability to underlie the storage and retrieval of genetic information. This Review highlights these recent developments as well as the applications enabled by the UBPs, including the expansion of the evolution process to include new functionality and the creation of semi-synthetic life that stores increased information.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVSjtLrL&md5=56b77b89dbbf47428bf446e8de15ca82
  4. 4
    Hirao, I.; Kimoto, M.; Yamashige, R. Acc. Chem. Res. 2012, 45, 2055– 2065 DOI: 10.1021/ar200257x
    Google Scholar
    There is no corresponding record for this reference.
  5. 5
    Benner, S. A.; Karalkar, N. B.; Hoshika, S.; Laos, R.; Shaw, R. W.; Matsuura, M.; Fajardo, D.; Moussatche, P. Cold Spring Harbor Perspect. Biol. 2016, 8, a023770 DOI: 10.1101/cshperspect.a023770
    Google Scholar
    5
    Alternative Watson-Crick synthetic genetic systems
    Benner, Steven A.; Karalkar, Nilesh B.; Hoshika, Shuichi; Laos, Roberto; Shaw, Ryan W.; Matsuura, Mariko; Fajardo, Diego; Moussatche, Patricia
    Cold Spring Harbor Perspectives in Biology (2016), 8 (11), a023770/1-a023770/27CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
    In its "grand challenge" format in chem., "synthesis" as an activity sets out a goal that is substantially beyond current theor. and technol. capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discoveryand paradigm changes in waysthat anal. cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biol.: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural historyon Earth. The outcomes in technol. include new diagnostic toolsthat have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpslWktrY%253D&md5=db3376ae594ae9894e2901926bf65133
  6. 6
    Craig, J. E.; Zhang, Y.; Gallagher, M. P. Mol. Microbiol. 1994, 11, 1159– 1168 DOI: 10.1111/j.1365-2958.1994.tb00392.x
    Google Scholar
    6
    Cloning of the nupC gene of Escherichia coli encoding a nucleoside transport system, and identification of an adjacent insertion element, IS 186
    Craig, Jane E.; Zhang, Yianbiao; Gallagher, Maurice P.
    Molecular Microbiology (1994), 11 (6), 1159-68CODEN: MOMIEE; ISSN:0950-382X.
    Escherichia coli is known to contain more than one active transport system for nucleoside uptake. In the present study the authors report the sequence of a gene encoding a 2nd nucleoside transport system, nupC (in addn. to nupG). An open reading frame (ORF) of 1200 bp was identified that codes for a hydrophobic polypeptide of 43,560 Da and an NupC fusion protein was shown to be membrane assocd. The native NupC protein is also identified, following over-expression. NupC exhibits short regions of homol. to several membrane-assocd. proteins, including LacY and Cyd. Anal. of the nupC promoter region revealed the presence of at least 2 putative CRP-binding sites, centered at -40 bp and -89 bp, which probably flank a CytR-binding site. In addn., an adjacent IS 186 element was identified and found to reside within a putative terminator structure, down-stream from the nupC ORF. This arrangement is shown to reflect the previously established gene order on the E. coli chromosome.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXksFWjs7w%253D&md5=346a6d5af71b3d9dfcd96ae49b27108c
  7. 7
    Norholm, M. H.; Dandanell, G. J. Bacteriol. 2001, 183, 4900– 4904 DOI: 10.1128/JB.183.16.4900-4904.2001
    Google Scholar
    7
    Specificity and topology of the Escherichia coli xanthosine permease, a representative of the NHS subfamily of the major facilitator superfamily
    Norholm, Morten H. H.; Dandanell, Gert
    Journal of Bacteriology (2001), 183 (16), 4900-4904CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    The specificity of XapB permease was compared with that of the known nucleoside transporters NupG and NupC. XapB-mediated xanthosine uptake is abolished by 2,4-dinitrophenol and exhibits satn. kinetics with an apparent Km of 136 μM. A 12-transmembrane-segment model was confirmed by translational fusions to alk. phosphatase and the α fragment of β-galactosidase.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXlslKktLo%253D&md5=0a6a5087400d8dc29a1aa240547445d9
  8. 8
    Westh Hansen, S. E.; Jensen, N.; Munch-Petersen, A. Eur. J. Biochem. 1987, 168, 385– 391 DOI: 10.1111/j.1432-1033.1987.tb13431.x
    Google Scholar
    8
    Studies on the sequence and structure of the Escherichia coli K-12 nupG gene, encoding a nucleoside-transport system
    Westh Hansen, Svend Erik; Jensen, Nina; Munch-Petersen, Agnete
    European Journal of Biochemistry (1987), 168 (2), 385-91CODEN: EJBCAI; ISSN:0014-2956.
    The nupG gene, encoding 1 of the 2 active nucleoside-transport systems in E. coli K-12, was cloned on the multicopy plasmid pBR322 and derivs. thereof. The recombinant plasmids complemented a chromosomal nupG mutation. A genetic map was detd. by digestion with restriction endonucleases and the nucleotide sequence of a 3-kb stretch of DNA was detd. on fragments cloned into M13 phages. An open reading frame of 1254 bp, encoding a protein with a calcd. mol. mass of 45.333 kDa, was deduced to be the coding region of nupG. Minicell-forming strains carrying plasmids contg. this gene produced a hydrophobic, membrane-bound polypeptide with an apparent mol. mass of approx. 43 kDa.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXltVWksQ%253D%253D&md5=8e1a9c4b46ca1a82b030285ff5e6d74f
  9. 9
    Ye, J.; van den Berg, B. EMBO J. 2004, 23, 3187– 3195 DOI: 10.1038/sj.emboj.7600330
    Google Scholar
    There is no corresponding record for this reference.
  10. 10
    Henderson, J. F.; Paterson, A. R. P. Nucleotide Metabolism: An Introduction; Academic Press: New York, 1973.
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Sandrini, M. P.; Piskur, J. Trends Biochem. Sci. 2005, 30, 225– 228 DOI: 10.1016/j.tibs.2005.03.003
    Google Scholar
    There is no corresponding record for this reference.
  12. 12
    Konrad, A.; Yarunova, E.; Tinta, T.; Piskur, J.; Liberles, D. A. Gene 2012, 492, 117– 120 DOI: 10.1016/j.gene.2011.10.039
    Google Scholar
    There is no corresponding record for this reference.
  13. 13
    Sivakumar, S.; Porter-Goff, M.; Patel, P. K.; Benoit, K.; Rhind, N. Methods 2004, 33, 213– 219 DOI: 10.1016/j.ymeth.2003.11.016
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Vernis, L.; Piskur, J.; Diffley, J. F. Nucleic Acids Res. 2003, 31, e120 DOI: 10.1093/nar/gng121
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Yan, H.; Tsai, M. D. Adv. Enzymol. Relat. Areas Mol. Biol. 1999, 73, 103– 143 DOI: 10.1002/9780470123195.ch4
    Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Sandrini, M. P.; Clausen, A. R.; On, S. L.; Aarestrup, F. M.; Munch-Petersen, B.; Piskur, J. J. Antimicrob. Chemother. 2007, 60, 510– 520 DOI: 10.1093/jac/dkm240
    Google Scholar
    16
    Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner
    Sandrini, Michael P. B.; Clausen, Anders R.; On, Stephen L. W.; Aarestrup, Frank M.; Munch-Petersen, Birgitte; Piskur, Jure
    Journal of Antimicrobial Chemotherapy (2007), 60 (3), 510-520CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press)
    Objectives: To investigate the bactericidal activity of antiviral and anticancer nucleoside analogs against a variety of pathogenic bacteria and characterize the activating enzymes, deoxyribonucleoside kinases (dNKs). Methods: Several FDA-approved nucleoside analog drugs were screened for their potential bactericidal activity against several clin. bacterial isolates and type strains. The authors identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes. These genes were tested for their ability to increase the susceptibility of a dNK-deficient E. coli strain to various analogs. The authors overexpressed, purified and characterized the substrate specificity and kinetic properties of the recombinant enzymes from S. enterica and B. cereus. Results: The tested Gram-neg. bacteria were susceptible to 3'-azido-3'-deoxythymidine (AZT) in the concn. range 0.03231.6 μM except for a single E. coli isolate and two Pseudomonas aeruginosa isolates which were resistant to the tested AZT concns. Purified recombinant S. enterica thymidine kinase phosphorylated AZT efficiently with a Km of 73.3 μM and kcat/Km of 6.6 × 104 s-1 M-1 and is the activator of this drug in vivo. 2',2'-Difluoro-2'-deoxycytidine (gemcitabine) was a potent antibiotic against Gram-pos. bacteria in the concn. range between 0.001 and 1.0 μM. The B. cereus deoxyadenosine kinase had a Km for gemcitabine of 33.5 μM and kcat/Km of 5.1 × 103 s-1 M-1 and activates gemcitabine in vivo. S. enterica and B. cereus are now amongst the first bacteria with a completely characterized set of dNK enzymes. Conclusions: Bacterial dNKs efficiently activate nucleoside analogs in a species-specific manner. Therefore, nucleoside analogs have a potential to be employed as antibiotics in the fight against emerging multiresistant bacteria.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXpt1Cmu7c%253D&md5=6b4f45be7b63ffb0fbf3ccb5f79662ab
  17. 17
    Wu, Y.; Fa, M.; Tae, E. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2002, 124, 14626– 14630 DOI: 10.1021/ja028050m
    Google Scholar
    There is no corresponding record for this reference.
  18. 18
    Matsuura, M. F.; Winiger, C. B.; Shaw, R. W.; Kim, M. J.; Kim, M. S.; Daugherty, A. B.; Chen, F.; Moussatche, P.; Moses, J. D.; Lutz, S.; Benner, S. A. ACS Synth. Biol. 2017, 6, 388– 394 DOI: 10.1021/acssynbio.6b00228
    Google Scholar
    18
    A Single Deoxynucleoside Kinase Variant from Drosophila melanogaster Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System
    Matsuura, Mariko F.; Winiger, Christian B.; Shaw, Ryan W.; Kim, Myong-Jung; Kim, Myong-Sang; Daugherty, Ashley B.; Chen, Fei; Moussatche, Patricia; Moses, Jennifer D.; Lutz, Stefan; Benner, Steven A.
    ACS Synthetic Biology (2017), 6 (3), 388-394CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Deoxynucleoside kinase from D. melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidines more efficiently than natural purine nucleosides, it accepts all four 2'-deoxynucleosides and many analogs, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS). By shuffling hydrogen bonding groups on the nucleobases, AEGIS adds potentially as many as four addnl. nucleobase pairs to the genetic "alphabet". Specifically, we show that DmdNK Q81E creates the monophosphates from the AEGIS nucleosides dP, dZ, dX, and dK (resp. 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone, dZ; 8-(β-D-2'-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1'-β-D-2'-deoxyribofuranosyl)-pyrimidine, dK). Using a coupled enzyme assay, in vitro kinetic parameters were obtained for three of these (dP, dX, and dK; the UV absorbance of dZ made it impossible to get its precise kinetic parameters). Thus, DmdNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2'-deoxynucleoside triphosphates in vitro and, perhaps, in vivo, in a cell able to manage plasmids contg. AEGIS DNA.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVajtrnJ&md5=3243e2181ea0f219ae975cbf06abe066
  19. 19
    Black, M. E.; Newcomb, T. G.; Wilson, H. M.; Loeb, L. A. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 3525– 3529 DOI: 10.1073/pnas.93.8.3525
    Google Scholar
    There is no corresponding record for this reference.
  20. 20
    Johansson, M.; van Rompay, A. R.; Degreve, B.; Balzarini, J.; Karlsson, A. J. Biol. Chem. 1999, 274, 23814– 23819 DOI: 10.1074/jbc.274.34.23814
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Munch-Petersen, A.; Jensen, N. Eur. J. Biochem. 1990, 190, 547– 551 DOI: 10.1111/j.1432-1033.1990.tb15608.x
    Google Scholar
    21
    Analysis of the regulatory region of the Escherichia coli nupG gene, encoding a nucleoside-transport protein
    Munch-Petersen, Agnete; Jensen, Nina
    European Journal of Biochemistry (1990), 190 (3), 547-51CODEN: EJBCAI; ISSN:0014-2956.
    The S1 mapping procedure when applied to an 800-bp fragment upstream of the mapG structural gene of Escherichia coli revealed 1 transcription initiation site. The corresponding promoter is neg. regulated by the cytR and the deoR repressors. The expression of the gene is activated by the complex between cAMP and its receptor protein. In strains lacking cAMP, the promoter expression is reduced, and it is no longer regulated by the cytR repressor, whereas the deoR regulation is retained.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXktVWhurc%253D&md5=0d46a27d3e085dd6de711e137bf8d228
  22. 22
    Yoshioka, A.; Tanaka, S.; Hiraoka, O.; Koyama, Y.; Hirota, Y.; Ayusawa, D.; Seno, T.; Garrett, C.; Wataya, Y. J. Biol. Chem. 1987, 262, 8235– 8241
    Google Scholar
    There is no corresponding record for this reference.
  23. 23
    Chou, I. N.; Zeiger, J.; Rapaport, E. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 2401– 2405 DOI: 10.1073/pnas.81.8.2401
    Google Scholar
    There is no corresponding record for this reference.
  24. 24
    Malyshev, D. A.; Dhami, K.; Lavergne, T.; Chen, T.; Dai, N.; Foster, J. M.; Correa, I. R., Jr.; Romesberg, F. E. Nature 2014, 509, 385– 388 DOI: 10.1038/nature13314
    Google Scholar
    24
    A semi-synthetic organism with an expanded genetic alphabet
    Malyshev, 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.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXotVyqtb8%253D&md5=97b4b184cda52cc809b1705e5e88ad8e
  25. 25
    Hantke, K. FEBS Lett. 1976, 70, 109– 112 DOI: 10.1016/0014-5793(76)80737-5
    Google Scholar
    There is no corresponding record for this reference.
  26. 26
    McKeown, M.; Kahn, M.; Hanawalt, P. J. Bacteriol. 1976, 126, 814– 822
    Google Scholar
    26
    Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport
    McKeown, Michael; Kahn, Michael; Hanawalt, Philip
    Journal of Bacteriology (1976), 126 (2), 814-22CODEN: JOBAAY; ISSN:0021-9193.
    A commonly used strain of Escherichia coli K-12 was shown to be deficient in the transport of a no. of nucleosides, including thymidine. Thymine incorporation was unaffected. Strain AB2497 exhibited a strikingly lower thymidine pulse-label incorporation at low (<1 μg/ml) thymidine concns. than do many other strains. The deficiency appeared to be due to mutation in a single gene. This gene, nup, is located at 10-13 min on the E. coli linkage map. In nup+ strains, the transport of a given nucleoside was relatively insensitive to large excesses of other nucleosides but was competitively inhibited by the same nucleoside. Mutants deficient in thymidine kinase are deficient in thymidine uptake but normal in deoxyadenosine uptake. A 2-step model for nucleoside transport is presented in which the first step, utilizing the nup gene product, is a nonspecific translocation of nucleoside to the interior of the cell. In the second step, the individual nucleosides are modified by cellular enzymes (e.g., nucleoside kinases) to facilitate accumulation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XksVels74%253D&md5=1688ab97da8659fd254a88284a9cbb0a
  27. 27
    Acimovic, Y.; Coe, I. R. Mol. Biol. Evol. 2002, 19, 2199– 2210 DOI: 10.1093/oxfordjournals.molbev.a004044
    Google Scholar
    There is no corresponding record for this reference.
  28. 28
    Zhang, Y.; Lamb, B.; Feldman, A. W.; Zhou, A. X.; Lavergne, T.; Li, L.; Romesberg, F. E. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 1317– 1322 DOI: 10.1073/pnas.1616443114
    Google Scholar
    There is no corresponding record for this reference.
  29. 29
    Zhang, 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. Nature 2017, 551, 644– 647 DOI: 10.1038/nature24659
    Google Scholar
    29
    A semi-synthetic organism that stores and retrieves increased genetic information
    Zhang, 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.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWhtL7L&md5=d04861904783b1a852d4089b28f09acf
  30. 30
    Ast, M.; Gruber, A.; Schmitz-Esser, S.; Neuhaus, H. E.; Kroth, P. G.; Horn, M.; Haferkamp, I. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 3621– 3626 DOI: 10.1073/pnas.0808862106
    Google Scholar
    There is no corresponding record for this reference.
  31. 31
    Nabel, C. S.; Manning, S. A.; Kohli, R. M. ACS Chem. Biol. 2012, 7, 20– 30 DOI: 10.1021/cb2002895
    Google Scholar
    There is no corresponding record for this reference.
  32. 32
    Sanchez-Romero, M. A.; Cota, I.; Casadesus, J. Curr. Opin. Microbiol. 2015, 25, 9– 16 DOI: 10.1016/j.mib.2015.03.004
    Google Scholar
    32
    DNA methylation in bacteria: from the methyl group to the methylome
    Sanchez-Romero, Maria A.; Cota, Ignacio; Casadesus, Josep
    Current Opinion in Microbiology (2015), 25 (), 9-16CODEN: COMIF7; ISSN:1369-5274. (Elsevier Ltd.)
    A review. Formation of C5-methyl-cytosine, N4-methyl-cytosine, and N6-methyl-adenine in bacterial genomes is postreplicative, and occurs at specific targets. Base methylation can modulate the interaction of DNA-binding proteins with their cognate sites, and controls chromosome replication, correction of DNA mismatches, cell cycle-coupled transcription, and formation of epigenetic lineages by phase variation. During four decades, the roles of DNA methylation in bacterial physiol. have been investigated by analyzing the contribution of individual Me groups or small Me group clusters to the control of DNA-protein interactions. Nowadays, single-mol. real-time sequencing can analyze the DNA methylation of the entire genome (the 'methylome'). Bacterial methylomes provide a wealth of information on the methylation marks present in bacterial genomes, and may open a new era in bacterial epigenomics.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXks1arsb0%253D&md5=6cb3d22fe59ad8e79b4406db8b733672
  33. 33
    Munzel, M.; Globisch, D.; Carell, T. Angew. Chem., Int. Ed. 2011, 50, 6460– 6468 DOI: 10.1002/anie.201101547
    Google Scholar
    There is no corresponding record for this reference.
  34. 34
    Henry, A. A.; Olsen, A. G.; Matsuda, S.; Yu, C.; Geierstanger, B. H.; Romesberg, F. E. J. Am. Chem. Soc. 2004, 126, 6923– 6931 DOI: 10.1021/ja049961u
    Google Scholar
    There is no corresponding record for this reference.
  35. 35
    Leconte, A. M.; Hwang, G. T.; Matsuda, S.; Capek, P.; Hari, Y.; Romesberg, F. E. J. Am. Chem. Soc. 2008, 130, 2336– 2343 DOI: 10.1021/ja078223d
    Google Scholar
    There is no corresponding record for this reference.
  36. 36
    Li, L.; Degardin, M.; Lavergne, T.; Malyshev, D. A.; Dhami, K.; Ordoukhanian, P.; Romesberg, F. E. J. Am. Chem. Soc. 2014, 136, 826– 829 DOI: 10.1021/ja408814g
    Google Scholar
    There is no corresponding record for this reference.
  37. 37
    Seo, Y. J.; Hwang, G. T.; Ordoukhanian, P.; Romesberg, F. E. J. Am. Chem. Soc. 2009, 131, 3246– 3252 DOI: 10.1021/ja807853m
    Google Scholar
    There is no corresponding record for this reference.
  38. 38
    Matsuda, S.; Henry, A. A.; Romesberg, F. E. J. Am. Chem. Soc. 2006, 128, 6369– 6375 DOI: 10.1021/ja057575m
    Google Scholar
    There is no corresponding record for this reference.
  39. 39
    Meyer, A. S.; Blandino, M.; Spratt, T. E. J. Biol. Chem. 2004, 279, 33043– 33046 DOI: 10.1074/jbc.C400232200
    Google Scholar
    There is no corresponding record for this reference.
  40. 40
    Morales, J. C.; Kool, E. T. J. Am. Chem. Soc. 1999, 121, 2323– 2324 DOI: 10.1021/ja983502+
    Google Scholar
    There is no corresponding record for this reference.
  41. 41
    Spratt, T. E. Biochemistry 2001, 40, 2647– 2652 DOI: 10.1021/bi002641c
    Google Scholar
    There is no corresponding record for this reference.
  42. 42
    Seo, Y. J.; Matsuda, S.; Romesberg, F. E. J. Am. Chem. Soc. 2009, 131, 5046– 5047 DOI: 10.1021/ja9006996
    Google Scholar
    There is no corresponding record for this reference.
  43. 43
    Sau, S. P.; Fahmi, N. E.; Liao, J. Y.; Bala, S.; Chaput, J. C. J. Org. Chem. 2016, 81, 2302– 2307 DOI: 10.1021/acs.joc.5b02768
    Google Scholar
    There is no corresponding record for this reference.
  44. 44
    Sau, S. P.; Chaput, J. C. Org. Lett. 2017, 19, 4379– 4382 DOI: 10.1021/acs.orglett.7b02099
    Google Scholar
    There is no corresponding record for this reference.
  45. 45
    Chaput, J. C.; Yu, H.; Zhang, S. Chem. Biol. 2012, 19, 1360– 1371 DOI: 10.1016/j.chembiol.2012.10.011
    Google Scholar
    45
    The emerging world of synthetic genetics
    Chaput, John C.; Yu, Hanyang; Zhang, Su
    Chemistry & Biology (Oxford, United Kingdom) (2012), 19 (11), 1360-1371CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
    A review. For over 20 years, labs. around the world have been applying the principles of Darwinian evolution to isolate DNA and RNA mols. with specific ligand-binding or catalytic activities. This area of synthetic biol., commonly referred to as in vitro genetics, is made possible by the availability of natural polymerases that can replicate genetic information in the lab. Moving beyond natural nucleic acids requires org. chem. to synthesize unnatural analogs and polymerase engineering to create enzymes that recognize artificial substrates. Progress in both of these areas has led to the emerging field of synthetic genetics, which explores the structural and functional properties of synthetic genetic polymers by in vitro evolution. This review examines recent advances in the Darwinian evolution of artificial genetic polymers and their potential downstream applications in exobiol., mol. medicine, and synthetic biol.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhslaqurfN&md5=110001564da8774878aca0de932e6e32
  46. 46
    Pinheiro, V. B.; Taylor, A. I.; Cozens, C.; Abramov, M.; Renders, M.; Zhang, S.; Chaput, J. C.; Wengel, J.; Peak-Chew, S. Y.; McLaughlin, S. H.; Herdewijn, P.; Holliger, P. Science 2012, 336, 341– 344 DOI: 10.1126/science.1217622
    Google Scholar
    There is no corresponding record for this reference.
  47. 47
    Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.; Martinek, V.; Xiang, Y.; Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; Florian, J.; Warshel, A.; Goodman, M. F. Biochemistry 2007, 46, 461– 471 DOI: 10.1021/bi061517b
    Google Scholar
    There is no corresponding record for this reference.
  48. 48
    Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.; Wilson, S. H.; Florian, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. Biochemistry 2008, 47, 870– 879 DOI: 10.1021/bi7014162
    Google Scholar
    There is no corresponding record for this reference.
  49. 49
    Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T. G.; Goodman, M. F.; McKenna, C. E. J. Am. Chem. Soc. 2010, 132, 7617– 7625 DOI: 10.1021/ja909370k
    Google Scholar
    There is no corresponding record for this reference.
  50. 50
    Oertell, K.; Chamberlain, B. T.; Wu, Y.; Ferri, E.; Kashemirov, B. A.; Beard, W. A.; Wilson, S. H.; McKenna, C. E.; Goodman, M. F. Biochemistry 2014, 53, 1842– 1848 DOI: 10.1021/bi500101z
    Google Scholar
    There is no corresponding record for this reference.
  51. 51
    Feldman, A. W.; Dien, V. T.; Romesberg, F. E. J. Am. Chem. Soc. 2017, 139, 2464– 2467 DOI: 10.1021/jacs.6b12731
    Google Scholar
    There is no corresponding record for this reference.
  52. 52
    Rabinowitz, J. D.; Kimball, E. Anal. Chem. 2007, 79, 6167– 6173 DOI: 10.1021/ac070470c
    Google Scholar
    There is no corresponding record for this reference.
  53. 53
    Manoharan, M.; Akinc, A.; Pandey, R. K.; Qin, J.; Hadwiger, P.; John, M.; Mills, K.; Charisse, K.; Maier, M. A.; Nechev, L.; Greene, E. M.; Pallan, P. S.; Rozners, E.; Rajeev, K. G.; Egli, M. Angew. Chem., Int. Ed. 2011, 50, 2284– 2288 DOI: 10.1002/anie.201006519
    Google Scholar
    There is no corresponding record for this reference.
  54. 54
    Mehta, A. P.; Li, H.; Reed, S. A.; Supekova, L.; Javahishvili, T.; Schultz, P. G. J. Am. Chem. Soc. 2016, 138, 7272– 7275 DOI: 10.1021/jacs.6b03904
    Google Scholar
    There is no corresponding record for this reference.
  55. 55
    Mehta, A. P.; Li, H.; Reed, S. A.; Supekova, L.; Javahishvili, T.; Schultz, P. G. J. Am. Chem. Soc. 2016, 138, 14230– 14233 DOI: 10.1021/jacs.6b09661
    Google Scholar
    There is no corresponding record for this reference.
  56. 56
    Nainar, S.; Beasley, S.; Fazio, M.; Kubota, M.; Dai, N.; Correa, I. R., Jr.; Spitale, R. C. ChemBioChem 2016, 17, 2149– 2152 DOI: 10.1002/cbic.201600300
    Google Scholar
    There is no corresponding record for this reference.
  57. 57
    Pezo, V.; Liu, F. W.; Abramov, M.; Froeyen, M.; Herdewijn, P.; Marliere, P. Angew. Chem., Int. Ed. 2013, 52, 8139– 8143 DOI: 10.1002/anie.201303288
    Google Scholar
    There is no corresponding record for this reference.
  58. 58
    Marliere, P.; Patrouix, J.; Doring, V.; Herdewijn, P.; Tricot, S.; Cruveiller, S.; Bouzon, M.; Mutzel, R. Angew. Chem., Int. Ed. 2011, 50, 7109– 7114 DOI: 10.1002/anie.201100535
    Google Scholar
    There is no corresponding record for this reference.
  59. 59
    Murthy, S.; Reddy, G. P. J. Cell. Physiol. 2006, 209, 711– 717 DOI: 10.1002/jcp.20842
    Google Scholar
    59
    Replitase: complete machinery for DNA synthesis
    Murthy, Shalini; Reddy, G. Prem-Veer
    Journal of Cellular Physiology (2006), 209 (3), 711-717CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Liss, Inc.)
    A review. Replication of nuclear DNA in eukaryotes presents a tremendous challenge, not only due to the size and complexity of the genome, but also because of the time constraint imposed by a limited duration of S-phase during which the entire genome has to be duplicated accurately and only once per cell division cycle. A challenge of this magnitude can only be met by the close coupling of DNA precursor synthesis to replication. Prokaryotic systems provide evidence for multienzyme and multiprotein complexes involved in DNA precursor synthesis and DNA replication. In addn., fractionation of nuclear proteins from proliferating mammalian cells shows co-sedimentation of enzymes involved in DNA replication with those required for synthesis of deoxyribonucleoside triphosphates (dNTPs). Such complexes can be isolated only from cells that are in the S-phase, but not from cells in the G0/G1-phases of the cell cycle. The kinetics of deoxyribonucleotide metab. supporting DNA replication in intact and permeabilized cells reveals close coupling and allosteric interaction between the enzymes of dNTP synthesis and DNA replication. These interactions contribute to channeling and compartmentation of deoxyribonucleotides in the microvicinity of DNA replication. A multienzyme and multiprotein megacomplex with these unique properties is called "replitase.". Here, the authors summarize some of the relevant evidence to date that supports the concept of replitase in mammalian cells, which originated from observations in the lab. of A. B. Pardee (G. P. Reddy and A. B. Pardee, 1980.). In addn., the authors show that androgen receptor (AR), which plays a crit. role in proliferation and viability of prostate cancer cells, is assocd. with replitase, and that identification of the constituents of replitase in androgen-dependent vs. androgen-independent prostate cancer cells may provide insights into androgen-regulated events that control proliferation of prostate cancer cells and potentially offer an effective strategy for the treatment of prostate cancer.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFOktLrI&md5=22356e76992b640ee798bec79c7e7aad
  60. 60
    Mathews, C. K. In Genetic Consequences of Nucleotide Pool Imbalance; de Serres, F. J., Ed.; Springer: Boston, MA, 1985.
    Google Scholar
    There is no corresponding record for this reference.

Cited By

Click to copy section linkSection link copied!
Citation Statements
  • Supporting
    Supporting0
  • Mentioning
    Mentioning36
  • Contrasting
    Contrasting0
Explore this article's citation statements on scite.ai
powered by  Scite

This article is cited by 36 publications.

  1. David Mueller, Remo Baettig, Tilmann Kuenzl, Emilio Rodríguez-Robles, Tania Michelle Roberts, Philippe Marlière, Sven Panke. Characterizing and Tailoring the Substrate Profile of a γ-Glutamyltransferase Variant. ACS Synthetic Biology 2024, Article ASAP.
  2. Chaoyang Zhu, Liang Xu, Longxin Chen, Zihan Zhang, Yuhan Zhang, Weiping Wu, Chengxiang Li, Shuang Liu, Shuqin Xiang, Shengwang Dai, Jay Zhang, Hui Guo, Yinjian Zhou, Feng Wang. Epitope-Directed Antibody Elicitation by Genetically Encoded Chemical Cross-Linking Reactivity in the Antigen. ACS Central Science 2023, 9 (6) , 1229-1240. https://doi.org/10.1021/acscentsci.3c00265
  3. Yubing Li, Clay Abraham, Oleg Suslov, Ozlem Yaren, Ryan W. Shaw, Myong-Jung Kim, Shuo Wan, Philippe Marliere, Steven A. Benner. Synthetic Biology Pathway to Nucleoside Triphosphates for Expanded Genetic Alphabets. ACS Synthetic Biology 2023, 12 (6) , 1772-1781. https://doi.org/10.1021/acssynbio.3c00060
  4. Verena N. Schreier, Morten O. Loehr, Evelyn Lattmann, Nathan W. Luedtke. Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo. ACS Chemical Biology 2022, 17 (7) , 1799-1810. https://doi.org/10.1021/acschembio.2c00153
  5. Koji Hashimoto, Emil C. Fischer, Floyd E. Romesberg. Efforts toward Further Integration of an Unnatural Base Pair into the Biology of a Semisynthetic Organism. Journal of the American Chemical Society 2021, 143 (23) , 8603-8607. https://doi.org/10.1021/jacs.1c03860
  6. Pouya Haratipour, Corinne Minard, Maryam Nakhjiri, Amirsoheil Negahbani, Brian T. Chamberlain, Jorge Osuna, Thomas G. Upton, Michelle Zhao, Boris A. Kashemirov, Charles E. McKenna. Completing the β,γ-CXY-dNTP Stereochemical Probe Toolkit: Synthetic Access to the dCTP Diastereomers and 31P and 19F NMR Correlations with Absolute Configurations. The Journal of Organic Chemistry 2020, 85 (22) , 14592-14609. https://doi.org/10.1021/acs.joc.0c01204
  7. Danyang Wang, Yu Zhang, Ralph E. Kleiner. Cell- and Polymerase-Selective Metabolic Labeling of Cellular RNA with 2′-Azidocytidine. Journal of the American Chemical Society 2020, 142 (34) , 14417-14421. https://doi.org/10.1021/jacs.0c04566
  8. Dehui Kong, Matina Movahedi, Yasaman Mahdavi-Amiri, Wayland Yeung, Tristan Tiburcio, Dickson Chen, Ryan Hili. Evolutionary Outcomes of Diversely Functionalized Aptamers Isolated from in Vitro Evolution. ACS Synthetic Biology 2020, 9 (1) , 43-52. https://doi.org/10.1021/acssynbio.9b00222
  9. Lei Wang, Bin Liu, Yuxue Liu, Yue Sun, Wujun Liu, Dayu Yu, Zongbao K. Zhao. Escherichia coli Strain Designed for Characterizing in Vivo Functions of Nicotinamide Adenine Dinucleotide Analogues. Organic Letters 2019, 21 (9) , 3218-3222. https://doi.org/10.1021/acs.orglett.9b00935
  10. Yu Zhang, Ralph E. Kleiner. A Metabolic Engineering Approach to Incorporate Modified Pyrimidine Nucleosides into Cellular RNA. Journal of the American Chemical Society 2019, 141 (8) , 3347-3351. https://doi.org/10.1021/jacs.8b11449
  11. Vivian T. Dien, Matthew Holcomb, Aaron W. Feldman, Emil C. Fischer, Tammy J. Dwyer, Floyd E. Romesberg. Progress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic Alphabet. Journal of the American Chemical Society 2018, 140 (47) , 16115-16123. https://doi.org/10.1021/jacs.8b08416
  12. Valérie Pezo, Camille Hassan, Dominique Louis, Bruno Sargueil, Piet Herdewijn, Philippe Marlière. Metabolic Recruitment and Directed Evolution of Nucleoside Triphosphate Uptake in Escherichia coli. ACS Synthetic Biology 2018, 7 (6) , 1565-1572. https://doi.org/10.1021/acssynbio.8b00048
  13. Jiahui Ma, Johanna Wehrle, Dennis Frank, Lina Lorenzen, Christoph Popp, Wolfgang Driever, Robert Grosse, Henning J. Jessen. Intracellular delivery and deep tissue penetration of nucleoside triphosphates using photocleavable covalently bound dendritic polycations. Chemical Science 2024, 15 (17) , 6478-6487. https://doi.org/10.1039/D3SC05669D
  14. Jie Zheng, Junjie Yang, Xu Liang, Mingliang Fang, Yulan Wang. Dual strategy for 13C-Metabolic flux analysis of central carbon and energy metabolism in Mammalian cells based on LC-isoMRM-MS. Talanta 2024, 266 , 125074. https://doi.org/10.1016/j.talanta.2023.125074
  15. Bianbian Huo, Chao Wang, Xiaoqi Hu, Honglei Wang, Gongming Zhu, Anlian Zhu, Lingjun Li. Peripheral substitution effects on unnatural base pairs: A case of brominated TPT3 to enhance replication fidelity. Bioorganic Chemistry 2023, 140 , 106827. https://doi.org/10.1016/j.bioorg.2023.106827
  16. Aude Blanchard, Mikhail Abramov, Camille Hassan, Philippe Marlière, Piet Herdewijn, Valérie Pezo. A microbiological system for screening the interference of XNA monomers with DNA and RNA metabolism. RSC Advances 2023, 13 (43) , 29862-29865. https://doi.org/10.1039/D3RA06172H
  17. 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
  18. Ralph E. Kleiner. Interrogating the transcriptome with metabolically incorporated ribonucleosides. Molecular Omics 2021, 17 (6) , 833-841. https://doi.org/10.1039/D1MO00334H
  19. Alan Costello, Ahmed H. Badran. Synthetic Biological Circuits within an Orthogonal Central Dogma. Trends in Biotechnology 2021, 39 (1) , 59-71. https://doi.org/10.1016/j.tibtech.2020.05.013
  20. Adeline Espinasse, Hannah K. Lembke, Angela A. Cao, Erin E. Carlson. Modified nucleoside triphosphates in bacterial research for in vitro and live-cell applications. RSC Chemical Biology 2020, 1 (5) , 333-351. https://doi.org/10.1039/D0CB00078G
  21. Nils Klöcker, Florian P. Weissenboeck, Andrea Rentmeister. Covalent labeling of nucleic acids. Chemical Society Reviews 2020, 49 (23) , 8749-8773. https://doi.org/10.1039/D0CS00600A
  22. Paola Handal-Marquez, Anupama Anupama, Valerie Pezo, Philippe Marlière, Piet Herdewijn, Vitor B. Pinheiro. Beneath the XNA world: Tools and targets to build novel biology. Current Opinion in Systems Biology 2020, 24 , 142-152. https://doi.org/10.1016/j.coisb.2020.10.013
  23. Karen Duffy, Sebastian Arangundy-Franklin, Philipp Holliger. Modified nucleic acids: replication, evolution, and next-generation therapeutics. BMC Biology 2020, 18 (1) https://doi.org/10.1186/s12915-020-00803-6
  24. Michiko Kimoto, Ichiro Hirao. Genetic alphabet expansion technology by creating unnatural base pairs. Chemical Society Reviews 2020, 49 (21) , 7602-7626. https://doi.org/10.1039/D0CS00457J
  25. Nils Muthmann, Katja Hartstock, Andrea Rentmeister. Chemo‐enzymatic treatment of RNA to facilitate analyses. WIREs RNA 2020, 11 (1) https://doi.org/10.1002/wrna.1561
  26. Zhenyu Zou, Wendi Xu, Chenyang Mi, Ying Xu, Ke Du, Bianbian Li, Yang Ye, Yihui Ling, Huidong Zhang. Ribonucleoside triphosphates promote T7 DNA replication and the lysis of T7-Infected Escherichia coli. Biochimie 2019, 167 , 25-33. https://doi.org/10.1016/j.biochi.2019.09.002
  27. Elena Eremeeva, Piet Herdewijn. Reprint of: Non Canonical Genetic Material. Current Opinion in Biotechnology 2019, 60 , 259-267. https://doi.org/10.1016/j.copbio.2019.11.009
  28. Ansgar Gruber, Ilka Haferkamp. Nucleotide Transport and Metabolism in Diatoms. Biomolecules 2019, 9 (12) , 761. https://doi.org/10.3390/biom9120761
  29. Fabienne Levi-Acobas, Adam Katolik, Pascal Röthlisberger, Thomas Cokelaer, Ivo Sarac, Masad J. Damha, Christian J. Leumann, Marcel Hollenstein. Compatibility of 5-ethynyl-2′F-ANA UTP with in vitro selection for the generation of base-modified, nuclease resistant aptamers. Organic & Biomolecular Chemistry 2019, 17 (35) , 8083-8087. https://doi.org/10.1039/C9OB01515A
  30. Elena Eremeeva, Piet Herdewijn. Non canonical genetic material. Current Opinion in Biotechnology 2019, 57 , 25-33. https://doi.org/10.1016/j.copbio.2018.12.001
  31. Marie Flamme, Luke K McKenzie, Ivo Sarac, Marcel Hollenstein. Chemical methods for the modification of RNA. Methods 2019, 161 , 64-82. https://doi.org/10.1016/j.ymeth.2019.03.018
  32. Floyd E. Romesberg. Synthetic Biology: The Chemist's Approach. Israel Journal of Chemistry 2019, 59 (1-2) , 91-94. https://doi.org/10.1002/ijch.201900014
  33. Pol Arranz-Gibert, Koen Vanderschuren, Farren J. Isaacs. Next-generation genetic code expansion. Current Opinion in Chemical Biology 2018, 46 , 203-211. https://doi.org/10.1016/j.cbpa.2018.07.020
  34. Vivian T Dien, Sydney E Morris, Rebekah J Karadeema, Floyd E Romesberg. Expansion of the genetic code via expansion of the genetic alphabet. Current Opinion in Chemical Biology 2018, 46 , 196-202. https://doi.org/10.1016/j.cbpa.2018.08.009
  35. Zbigniew Zawada, Ameneh Tatar, Pavle Mocilac, Miloš Buděšínský, Tomáš Kraus. Transport of Nucleoside Triphosphates into Cells by Artificial Molecular Transporters. Angewandte Chemie 2018, 130 (31) , 10039-10043. https://doi.org/10.1002/ange.201801306
  36. Zbigniew Zawada, Ameneh Tatar, Pavle Mocilac, Miloš Buděšínský, Tomáš Kraus. Transport of Nucleoside Triphosphates into Cells by Artificial Molecular Transporters. Angewandte Chemie International Edition 2018, 57 (31) , 9891-9895. https://doi.org/10.1002/anie.201801306
Download PDF
Open PDF
Go to Journal of the American Chemical Society
Get e-Alerts
Get e-Alerts

Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2018, 140, 4, 1447–1454
Click to copy citationCitation copied!
https://doi.org/10.1021/jacs.7b11404
Published January 17, 2018
Copyright © 2018 American Chemical Society
Request reuse permissions

Article Views

5285

Altmetric

8

Citations

36
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract 抽象

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1 图 1

    Figure 1. (A) Structure of nucleobase-modified nucleoside triphosphates used for the inhibition assay; sugar and phosphates omitted for clarity. (B) Percent inhibition of uptake of [α32P]ATP (50 μM) by the analogues indicated (500 μM each). Data shown are the average and SEM of three independent trials.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. (A) Structure of sugar modified nucleoside triphosphate analogues used for the inhibition assay. Base, one of the natural nucleobases; PPPO, triphosphate. (B) Percent inhibition of uptake of [α32P]ATP (50 μM) by the analogues indicated (500 μM each). Data shown are the average and SEM of three independent trials.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. (A) Structure of triphosphate-modified nucleoside triphosphates used for the inhibition assay. Base, one of the natural nucleobases. (B) Percent inhibition of uptake of [α32P]ATP (50 μM) by the analogues indicated (500 μM each). Data shown are the average and SEM of three independent trials.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. RNA and DNA radiolabeled with [α32P]-labeled analogues of each natural nucleotide.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 60 other publications.

    1. 1
      Chen, T.; Hongdilokkul, N.; Liu, Z.; Thirunavukarasu, D.; Romesberg, F. E. Curr. Opin. Chem. Biol. 2016, 34, 80– 87 DOI: 10.1016/j.cbpa.2016.08.001
      1
      The expanding world of DNA and RNA
      Chen, Tingjian; Hongdilokkul, Narupat; Liu, Zhixia; Thirunavukarasu, Deepak; Romesberg, Floyd E.
      Current Opinion in Chemical Biology (2016), 34 (), 80-87CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      DNA and RNA are remarkable because they can both encode information and possess desired properties, including the ability to bind specific targets or catalyze specific reactions. Nucleotide modifications that do not interfere with enzymic synthesis are now being used to bestow DNA or RNA with properties that further increase their utility, including phosphate and sugar modifications that increase nuclease resistance, nucleobase modifications that increase the range of activities possible, and even whole nucleobase replacement that results in selective pairing and the creation of unnatural base pairs that increase the information content. These modifications are increasingly being applied both in vitro and in vivo, including in efforts to create semi-synthetic organisms with altered or expanded genetic alphabets.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsVWmtr%252FO&md5=442970017f6d14ab44454a16bcb86303
    2. 2
      Houlihan, G.; Arangundy-Franklin, S.; Holliger, P. Acc. Chem. Res. 2017, 50, 1079– 1087 DOI: 10.1021/acs.accounts.7b00056
      There is no corresponding record for this reference.
    3. 3
      Malyshev, D. A.; Romesberg, F. E. Angew. Chem., Int. Ed. 2015, 54, 11930– 11944 DOI: 10.1002/anie.201502890
      3
      The expanded genetic alphabet
      Malyshev, Denis A.; Romesberg, Floyd E.
      Angewandte Chemie, International Edition (2015), 54 (41), 11930-11944CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. All biol. information, since the last common ancestor of all life on Earth, has been encoded by a genetic alphabet consisting of only four nucleotides that form two base pairs. Long-standing efforts to develop two synthetic nucleotides that form a third, unnatural base pair (UBP) have recently yielded three promising candidates, one based on alternative hydrogen bonding, and two based on hydrophobic and packing forces. All three of these UBPs are replicated and transcribed with remarkable efficiency and fidelity, and the latter two thus demonstrate that hydrogen bonding is not unique in its ability to underlie the storage and retrieval of genetic information. This Review highlights these recent developments as well as the applications enabled by the UBPs, including the expansion of the evolution process to include new functionality and the creation of semi-synthetic life that stores increased information.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsVSjtLrL&md5=56b77b89dbbf47428bf446e8de15ca82
    4. 4
      Hirao, I.; Kimoto, M.; Yamashige, R. Acc. Chem. Res. 2012, 45, 2055– 2065 DOI: 10.1021/ar200257x
      There is no corresponding record for this reference.
    5. 5
      Benner, S. A.; Karalkar, N. B.; Hoshika, S.; Laos, R.; Shaw, R. W.; Matsuura, M.; Fajardo, D.; Moussatche, P. Cold Spring Harbor Perspect. Biol. 2016, 8, a023770 DOI: 10.1101/cshperspect.a023770
      5
      Alternative Watson-Crick synthetic genetic systems
      Benner, Steven A.; Karalkar, Nilesh B.; Hoshika, Shuichi; Laos, Roberto; Shaw, Ryan W.; Matsuura, Mariko; Fajardo, Diego; Moussatche, Patricia
      Cold Spring Harbor Perspectives in Biology (2016), 8 (11), a023770/1-a023770/27CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
      In its "grand challenge" format in chem., "synthesis" as an activity sets out a goal that is substantially beyond current theor. and technol. capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discoveryand paradigm changes in waysthat anal. cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biol.: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural historyon Earth. The outcomes in technol. include new diagnostic toolsthat have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpslWktrY%253D&md5=db3376ae594ae9894e2901926bf65133
    6. 6
      Craig, J. E.; Zhang, Y.; Gallagher, M. P. Mol. Microbiol. 1994, 11, 1159– 1168 DOI: 10.1111/j.1365-2958.1994.tb00392.x
      6
      Cloning of the nupC gene of Escherichia coli encoding a nucleoside transport system, and identification of an adjacent insertion element, IS 186
      Craig, Jane E.; Zhang, Yianbiao; Gallagher, Maurice P.
      Molecular Microbiology (1994), 11 (6), 1159-68CODEN: MOMIEE; ISSN:0950-382X.
      Escherichia coli is known to contain more than one active transport system for nucleoside uptake. In the present study the authors report the sequence of a gene encoding a 2nd nucleoside transport system, nupC (in addn. to nupG). An open reading frame (ORF) of 1200 bp was identified that codes for a hydrophobic polypeptide of 43,560 Da and an NupC fusion protein was shown to be membrane assocd. The native NupC protein is also identified, following over-expression. NupC exhibits short regions of homol. to several membrane-assocd. proteins, including LacY and Cyd. Anal. of the nupC promoter region revealed the presence of at least 2 putative CRP-binding sites, centered at -40 bp and -89 bp, which probably flank a CytR-binding site. In addn., an adjacent IS 186 element was identified and found to reside within a putative terminator structure, down-stream from the nupC ORF. This arrangement is shown to reflect the previously established gene order on the E. coli chromosome.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXksFWjs7w%253D&md5=346a6d5af71b3d9dfcd96ae49b27108c
    7. 7
      Norholm, M. H.; Dandanell, G. J. Bacteriol. 2001, 183, 4900– 4904 DOI: 10.1128/JB.183.16.4900-4904.2001
      7
      Specificity and topology of the Escherichia coli xanthosine permease, a representative of the NHS subfamily of the major facilitator superfamily
      Norholm, Morten H. H.; Dandanell, Gert
      Journal of Bacteriology (2001), 183 (16), 4900-4904CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      The specificity of XapB permease was compared with that of the known nucleoside transporters NupG and NupC. XapB-mediated xanthosine uptake is abolished by 2,4-dinitrophenol and exhibits satn. kinetics with an apparent Km of 136 μM. A 12-transmembrane-segment model was confirmed by translational fusions to alk. phosphatase and the α fragment of β-galactosidase.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXlslKktLo%253D&md5=0a6a5087400d8dc29a1aa240547445d9
    8. 8
      Westh Hansen, S. E.; Jensen, N.; Munch-Petersen, A. Eur. J. Biochem. 1987, 168, 385– 391 DOI: 10.1111/j.1432-1033.1987.tb13431.x
      8
      Studies on the sequence and structure of the Escherichia coli K-12 nupG gene, encoding a nucleoside-transport system
      Westh Hansen, Svend Erik; Jensen, Nina; Munch-Petersen, Agnete
      European Journal of Biochemistry (1987), 168 (2), 385-91CODEN: EJBCAI; ISSN:0014-2956.
      The nupG gene, encoding 1 of the 2 active nucleoside-transport systems in E. coli K-12, was cloned on the multicopy plasmid pBR322 and derivs. thereof. The recombinant plasmids complemented a chromosomal nupG mutation. A genetic map was detd. by digestion with restriction endonucleases and the nucleotide sequence of a 3-kb stretch of DNA was detd. on fragments cloned into M13 phages. An open reading frame of 1254 bp, encoding a protein with a calcd. mol. mass of 45.333 kDa, was deduced to be the coding region of nupG. Minicell-forming strains carrying plasmids contg. this gene produced a hydrophobic, membrane-bound polypeptide with an apparent mol. mass of approx. 43 kDa.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXltVWksQ%253D%253D&md5=8e1a9c4b46ca1a82b030285ff5e6d74f
    9. 9
      Ye, J.; van den Berg, B. EMBO J. 2004, 23, 3187– 3195 DOI: 10.1038/sj.emboj.7600330
      There is no corresponding record for this reference.
    10. 10
      Henderson, J. F.; Paterson, A. R. P. Nucleotide Metabolism: An Introduction; Academic Press: New York, 1973.
      There is no corresponding record for this reference.
    11. 11
      Sandrini, M. P.; Piskur, J. Trends Biochem. Sci. 2005, 30, 225– 228 DOI: 10.1016/j.tibs.2005.03.003
      There is no corresponding record for this reference.
    12. 12
      Konrad, A.; Yarunova, E.; Tinta, T.; Piskur, J.; Liberles, D. A. Gene 2012, 492, 117– 120 DOI: 10.1016/j.gene.2011.10.039
      There is no corresponding record for this reference.
    13. 13
      Sivakumar, S.; Porter-Goff, M.; Patel, P. K.; Benoit, K.; Rhind, N. Methods 2004, 33, 213– 219 DOI: 10.1016/j.ymeth.2003.11.016
      There is no corresponding record for this reference.
    14. 14
      Vernis, L.; Piskur, J.; Diffley, J. F. Nucleic Acids Res. 2003, 31, e120 DOI: 10.1093/nar/gng121
      There is no corresponding record for this reference.
    15. 15
      Yan, H.; Tsai, M. D. Adv. Enzymol. Relat. Areas Mol. Biol. 1999, 73, 103– 143 DOI: 10.1002/9780470123195.ch4
      There is no corresponding record for this reference.
    16. 16
      Sandrini, M. P.; Clausen, A. R.; On, S. L.; Aarestrup, F. M.; Munch-Petersen, B.; Piskur, J. J. Antimicrob. Chemother. 2007, 60, 510– 520 DOI: 10.1093/jac/dkm240
      16
      Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner
      Sandrini, Michael P. B.; Clausen, Anders R.; On, Stephen L. W.; Aarestrup, Frank M.; Munch-Petersen, Birgitte; Piskur, Jure
      Journal of Antimicrobial Chemotherapy (2007), 60 (3), 510-520CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press)
      Objectives: To investigate the bactericidal activity of antiviral and anticancer nucleoside analogs against a variety of pathogenic bacteria and characterize the activating enzymes, deoxyribonucleoside kinases (dNKs). Methods: Several FDA-approved nucleoside analog drugs were screened for their potential bactericidal activity against several clin. bacterial isolates and type strains. The authors identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes. These genes were tested for their ability to increase the susceptibility of a dNK-deficient E. coli strain to various analogs. The authors overexpressed, purified and characterized the substrate specificity and kinetic properties of the recombinant enzymes from S. enterica and B. cereus. Results: The tested Gram-neg. bacteria were susceptible to 3'-azido-3'-deoxythymidine (AZT) in the concn. range 0.03231.6 μM except for a single E. coli isolate and two Pseudomonas aeruginosa isolates which were resistant to the tested AZT concns. Purified recombinant S. enterica thymidine kinase phosphorylated AZT efficiently with a Km of 73.3 μM and kcat/Km of 6.6 × 104 s-1 M-1 and is the activator of this drug in vivo. 2',2'-Difluoro-2'-deoxycytidine (gemcitabine) was a potent antibiotic against Gram-pos. bacteria in the concn. range between 0.001 and 1.0 μM. The B. cereus deoxyadenosine kinase had a Km for gemcitabine of 33.5 μM and kcat/Km of 5.1 × 103 s-1 M-1 and activates gemcitabine in vivo. S. enterica and B. cereus are now amongst the first bacteria with a completely characterized set of dNK enzymes. Conclusions: Bacterial dNKs efficiently activate nucleoside analogs in a species-specific manner. Therefore, nucleoside analogs have a potential to be employed as antibiotics in the fight against emerging multiresistant bacteria.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXpt1Cmu7c%253D&md5=6b4f45be7b63ffb0fbf3ccb5f79662ab
    17. 17
      Wu, Y.; Fa, M.; Tae, E. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2002, 124, 14626– 14630 DOI: 10.1021/ja028050m
      There is no corresponding record for this reference.
    18. 18
      Matsuura, M. F.; Winiger, C. B.; Shaw, R. W.; Kim, M. J.; Kim, M. S.; Daugherty, A. B.; Chen, F.; Moussatche, P.; Moses, J. D.; Lutz, S.; Benner, S. A. ACS Synth. Biol. 2017, 6, 388– 394 DOI: 10.1021/acssynbio.6b00228
      18
      A Single Deoxynucleoside Kinase Variant from Drosophila melanogaster Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System
      Matsuura, Mariko F.; Winiger, Christian B.; Shaw, Ryan W.; Kim, Myong-Jung; Kim, Myong-Sang; Daugherty, Ashley B.; Chen, Fei; Moussatche, Patricia; Moses, Jennifer D.; Lutz, Stefan; Benner, Steven A.
      ACS Synthetic Biology (2017), 6 (3), 388-394CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Deoxynucleoside kinase from D. melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidines more efficiently than natural purine nucleosides, it accepts all four 2'-deoxynucleosides and many analogs, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS). By shuffling hydrogen bonding groups on the nucleobases, AEGIS adds potentially as many as four addnl. nucleobase pairs to the genetic "alphabet". Specifically, we show that DmdNK Q81E creates the monophosphates from the AEGIS nucleosides dP, dZ, dX, and dK (resp. 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone, dZ; 8-(β-D-2'-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1'-β-D-2'-deoxyribofuranosyl)-pyrimidine, dK). Using a coupled enzyme assay, in vitro kinetic parameters were obtained for three of these (dP, dX, and dK; the UV absorbance of dZ made it impossible to get its precise kinetic parameters). Thus, DmdNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2'-deoxynucleoside triphosphates in vitro and, perhaps, in vivo, in a cell able to manage plasmids contg. AEGIS DNA.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVajtrnJ&md5=3243e2181ea0f219ae975cbf06abe066
    19. 19
      Black, M. E.; Newcomb, T. G.; Wilson, H. M.; Loeb, L. A. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 3525– 3529 DOI: 10.1073/pnas.93.8.3525
      There is no corresponding record for this reference.
    20. 20
      Johansson, M.; van Rompay, A. R.; Degreve, B.; Balzarini, J.; Karlsson, A. J. Biol. Chem. 1999, 274, 23814– 23819 DOI: 10.1074/jbc.274.34.23814
      There is no corresponding record for this reference.
    21. 21
      Munch-Petersen, A.; Jensen, N. Eur. J. Biochem. 1990, 190, 547– 551 DOI: 10.1111/j.1432-1033.1990.tb15608.x
      21
      Analysis of the regulatory region of the Escherichia coli nupG gene, encoding a nucleoside-transport protein
      Munch-Petersen, Agnete; Jensen, Nina
      European Journal of Biochemistry (1990), 190 (3), 547-51CODEN: EJBCAI; ISSN:0014-2956.
      The S1 mapping procedure when applied to an 800-bp fragment upstream of the mapG structural gene of Escherichia coli revealed 1 transcription initiation site. The corresponding promoter is neg. regulated by the cytR and the deoR repressors. The expression of the gene is activated by the complex between cAMP and its receptor protein. In strains lacking cAMP, the promoter expression is reduced, and it is no longer regulated by the cytR repressor, whereas the deoR regulation is retained.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXktVWhurc%253D&md5=0d46a27d3e085dd6de711e137bf8d228
    22. 22
      Yoshioka, A.; Tanaka, S.; Hiraoka, O.; Koyama, Y.; Hirota, Y.; Ayusawa, D.; Seno, T.; Garrett, C.; Wataya, Y. J. Biol. Chem. 1987, 262, 8235– 8241
      There is no corresponding record for this reference.
    23. 23
      Chou, I. N.; Zeiger, J.; Rapaport, E. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 2401– 2405 DOI: 10.1073/pnas.81.8.2401
      There is no corresponding record for this reference.
    24. 24
      Malyshev, D. A.; Dhami, K.; Lavergne, T.; Chen, T.; Dai, N.; Foster, J. M.; Correa, I. R., Jr.; Romesberg, F. E. Nature 2014, 509, 385– 388 DOI: 10.1038/nature13314
      24
      A semi-synthetic organism with an expanded genetic alphabet
      Malyshev, 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.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXotVyqtb8%253D&md5=97b4b184cda52cc809b1705e5e88ad8e
    25. 25
      Hantke, K. FEBS Lett. 1976, 70, 109– 112 DOI: 10.1016/0014-5793(76)80737-5
      There is no corresponding record for this reference.
    26. 26
      McKeown, M.; Kahn, M.; Hanawalt, P. J. Bacteriol. 1976, 126, 814– 822
      26
      Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport
      McKeown, Michael; Kahn, Michael; Hanawalt, Philip
      Journal of Bacteriology (1976), 126 (2), 814-22CODEN: JOBAAY; ISSN:0021-9193.
      A commonly used strain of Escherichia coli K-12 was shown to be deficient in the transport of a no. of nucleosides, including thymidine. Thymine incorporation was unaffected. Strain AB2497 exhibited a strikingly lower thymidine pulse-label incorporation at low (<1 μg/ml) thymidine concns. than do many other strains. The deficiency appeared to be due to mutation in a single gene. This gene, nup, is located at 10-13 min on the E. coli linkage map. In nup+ strains, the transport of a given nucleoside was relatively insensitive to large excesses of other nucleosides but was competitively inhibited by the same nucleoside. Mutants deficient in thymidine kinase are deficient in thymidine uptake but normal in deoxyadenosine uptake. A 2-step model for nucleoside transport is presented in which the first step, utilizing the nup gene product, is a nonspecific translocation of nucleoside to the interior of the cell. In the second step, the individual nucleosides are modified by cellular enzymes (e.g., nucleoside kinases) to facilitate accumulation.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE28XksVels74%253D&md5=1688ab97da8659fd254a88284a9cbb0a
    27. 27
      Acimovic, Y.; Coe, I. R. Mol. Biol. Evol. 2002, 19, 2199– 2210 DOI: 10.1093/oxfordjournals.molbev.a004044
      There is no corresponding record for this reference.
    28. 28
      Zhang, Y.; Lamb, B.; Feldman, A. W.; Zhou, A. X.; Lavergne, T.; Li, L.; Romesberg, F. E. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 1317– 1322 DOI: 10.1073/pnas.1616443114
      There is no corresponding record for this reference.
    29. 29
      Zhang, 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. Nature 2017, 551, 644– 647 DOI: 10.1038/nature24659
      29
      A semi-synthetic organism that stores and retrieves increased genetic information
      Zhang, 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.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWhtL7L&md5=d04861904783b1a852d4089b28f09acf
    30. 30
      Ast, M.; Gruber, A.; Schmitz-Esser, S.; Neuhaus, H. E.; Kroth, P. G.; Horn, M.; Haferkamp, I. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 3621– 3626 DOI: 10.1073/pnas.0808862106
      There is no corresponding record for this reference.
    31. 31
      Nabel, C. S.; Manning, S. A.; Kohli, R. M. ACS Chem. Biol. 2012, 7, 20– 30 DOI: 10.1021/cb2002895
      There is no corresponding record for this reference.
    32. 32
      Sanchez-Romero, M. A.; Cota, I.; Casadesus, J. Curr. Opin. Microbiol. 2015, 25, 9– 16 DOI: 10.1016/j.mib.2015.03.004
      32
      DNA methylation in bacteria: from the methyl group to the methylome
      Sanchez-Romero, Maria A.; Cota, Ignacio; Casadesus, Josep
      Current Opinion in Microbiology (2015), 25 (), 9-16CODEN: COMIF7; ISSN:1369-5274. (Elsevier Ltd.)
      A review. Formation of C5-methyl-cytosine, N4-methyl-cytosine, and N6-methyl-adenine in bacterial genomes is postreplicative, and occurs at specific targets. Base methylation can modulate the interaction of DNA-binding proteins with their cognate sites, and controls chromosome replication, correction of DNA mismatches, cell cycle-coupled transcription, and formation of epigenetic lineages by phase variation. During four decades, the roles of DNA methylation in bacterial physiol. have been investigated by analyzing the contribution of individual Me groups or small Me group clusters to the control of DNA-protein interactions. Nowadays, single-mol. real-time sequencing can analyze the DNA methylation of the entire genome (the 'methylome'). Bacterial methylomes provide a wealth of information on the methylation marks present in bacterial genomes, and may open a new era in bacterial epigenomics.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXks1arsb0%253D&md5=6cb3d22fe59ad8e79b4406db8b733672
    33. 33
      Munzel, M.; Globisch, D.; Carell, T. Angew. Chem., Int. Ed. 2011, 50, 6460– 6468 DOI: 10.1002/anie.201101547
      There is no corresponding record for this reference.
    34. 34
      Henry, A. A.; Olsen, A. G.; Matsuda, S.; Yu, C.; Geierstanger, B. H.; Romesberg, F. E. J. Am. Chem. Soc. 2004, 126, 6923– 6931 DOI: 10.1021/ja049961u
      There is no corresponding record for this reference.
    35. 35
      Leconte, A. M.; Hwang, G. T.; Matsuda, S.; Capek, P.; Hari, Y.; Romesberg, F. E. J. Am. Chem. Soc. 2008, 130, 2336– 2343 DOI: 10.1021/ja078223d
      There is no corresponding record for this reference.
    36. 36
      Li, L.; Degardin, M.; Lavergne, T.; Malyshev, D. A.; Dhami, K.; Ordoukhanian, P.; Romesberg, F. E. J. Am. Chem. Soc. 2014, 136, 826– 829 DOI: 10.1021/ja408814g
      There is no corresponding record for this reference.
    37. 37
      Seo, Y. J.; Hwang, G. T.; Ordoukhanian, P.; Romesberg, F. E. J. Am. Chem. Soc. 2009, 131, 3246– 3252 DOI: 10.1021/ja807853m
      There is no corresponding record for this reference.
    38. 38
      Matsuda, S.; Henry, A. A.; Romesberg, F. E. J. Am. Chem. Soc. 2006, 128, 6369– 6375 DOI: 10.1021/ja057575m
      There is no corresponding record for this reference.
    39. 39
      Meyer, A. S.; Blandino, M.; Spratt, T. E. J. Biol. Chem. 2004, 279, 33043– 33046 DOI: 10.1074/jbc.C400232200
      There is no corresponding record for this reference.
    40. 40
      Morales, J. C.; Kool, E. T. J. Am. Chem. Soc. 1999, 121, 2323– 2324 DOI: 10.1021/ja983502+
      There is no corresponding record for this reference.
    41. 41
      Spratt, T. E. Biochemistry 2001, 40, 2647– 2652 DOI: 10.1021/bi002641c
      There is no corresponding record for this reference.
    42. 42
      Seo, Y. J.; Matsuda, S.; Romesberg, F. E. J. Am. Chem. Soc. 2009, 131, 5046– 5047 DOI: 10.1021/ja9006996
      There is no corresponding record for this reference.
    43. 43
      Sau, S. P.; Fahmi, N. E.; Liao, J. Y.; Bala, S.; Chaput, J. C. J. Org. Chem. 2016, 81, 2302– 2307 DOI: 10.1021/acs.joc.5b02768
      There is no corresponding record for this reference.
    44. 44
      Sau, S. P.; Chaput, J. C. Org. Lett. 2017, 19, 4379– 4382 DOI: 10.1021/acs.orglett.7b02099
      There is no corresponding record for this reference.
    45. 45
      Chaput, J. C.; Yu, H.; Zhang, S. Chem. Biol. 2012, 19, 1360– 1371 DOI: 10.1016/j.chembiol.2012.10.011
      45
      The emerging world of synthetic genetics
      Chaput, John C.; Yu, Hanyang; Zhang, Su
      Chemistry & Biology (Oxford, United Kingdom) (2012), 19 (11), 1360-1371CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
      A review. For over 20 years, labs. around the world have been applying the principles of Darwinian evolution to isolate DNA and RNA mols. with specific ligand-binding or catalytic activities. This area of synthetic biol., commonly referred to as in vitro genetics, is made possible by the availability of natural polymerases that can replicate genetic information in the lab. Moving beyond natural nucleic acids requires org. chem. to synthesize unnatural analogs and polymerase engineering to create enzymes that recognize artificial substrates. Progress in both of these areas has led to the emerging field of synthetic genetics, which explores the structural and functional properties of synthetic genetic polymers by in vitro evolution. This review examines recent advances in the Darwinian evolution of artificial genetic polymers and their potential downstream applications in exobiol., mol. medicine, and synthetic biol.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhslaqurfN&md5=110001564da8774878aca0de932e6e32
    46. 46
      Pinheiro, V. B.; Taylor, A. I.; Cozens, C.; Abramov, M.; Renders, M.; Zhang, S.; Chaput, J. C.; Wengel, J.; Peak-Chew, S. Y.; McLaughlin, S. H.; Herdewijn, P.; Holliger, P. Science 2012, 336, 341– 344 DOI: 10.1126/science.1217622
      There is no corresponding record for this reference.
    47. 47
      Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.; Martinek, V.; Xiang, Y.; Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; Florian, J.; Warshel, A.; Goodman, M. F. Biochemistry 2007, 46, 461– 471 DOI: 10.1021/bi061517b
      There is no corresponding record for this reference.
    48. 48
      Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.; Wilson, S. H.; Florian, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. Biochemistry 2008, 47, 870– 879 DOI: 10.1021/bi7014162
      There is no corresponding record for this reference.
    49. 49
      Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T. G.; Goodman, M. F.; McKenna, C. E. J. Am. Chem. Soc. 2010, 132, 7617– 7625 DOI: 10.1021/ja909370k
      There is no corresponding record for this reference.
    50. 50
      Oertell, K.; Chamberlain, B. T.; Wu, Y.; Ferri, E.; Kashemirov, B. A.; Beard, W. A.; Wilson, S. H.; McKenna, C. E.; Goodman, M. F. Biochemistry 2014, 53, 1842– 1848 DOI: 10.1021/bi500101z
      There is no corresponding record for this reference.
    51. 51
      Feldman, A. W.; Dien, V. T.; Romesberg, F. E. J. Am. Chem. Soc. 2017, 139, 2464– 2467 DOI: 10.1021/jacs.6b12731
      There is no corresponding record for this reference.
    52. 52
      Rabinowitz, J. D.; Kimball, E. Anal. Chem. 2007, 79, 6167– 6173 DOI: 10.1021/ac070470c
      There is no corresponding record for this reference.
    53. 53
      Manoharan, M.; Akinc, A.; Pandey, R. K.; Qin, J.; Hadwiger, P.; John, M.; Mills, K.; Charisse, K.; Maier, M. A.; Nechev, L.; Greene, E. M.; Pallan, P. S.; Rozners, E.; Rajeev, K. G.; Egli, M. Angew. Chem., Int. Ed. 2011, 50, 2284– 2288 DOI: 10.1002/anie.201006519
      There is no corresponding record for this reference.
    54. 54
      Mehta, A. P.; Li, H.; Reed, S. A.; Supekova, L.; Javahishvili, T.; Schultz, P. G. J. Am. Chem. Soc. 2016, 138, 7272– 7275 DOI: 10.1021/jacs.6b03904
      There is no corresponding record for this reference.
    55. 55
      Mehta, A. P.; Li, H.; Reed, S. A.; Supekova, L.; Javahishvili, T.; Schultz, P. G. J. Am. Chem. Soc. 2016, 138, 14230– 14233 DOI: 10.1021/jacs.6b09661
      There is no corresponding record for this reference.
    56. 56
      Nainar, S.; Beasley, S.; Fazio, M.; Kubota, M.; Dai, N.; Correa, I. R., Jr.; Spitale, R. C. ChemBioChem 2016, 17, 2149– 2152 DOI: 10.1002/cbic.201600300
      There is no corresponding record for this reference.
    57. 57
      Pezo, V.; Liu, F. W.; Abramov, M.; Froeyen, M.; Herdewijn, P.; Marliere, P. Angew. Chem., Int. Ed. 2013, 52, 8139– 8143 DOI: 10.1002/anie.201303288
      There is no corresponding record for this reference.
    58. 58
      Marliere, P.; Patrouix, J.; Doring, V.; Herdewijn, P.; Tricot, S.; Cruveiller, S.; Bouzon, M.; Mutzel, R. Angew. Chem., Int. Ed. 2011, 50, 7109– 7114 DOI: 10.1002/anie.201100535
      There is no corresponding record for this reference.
    59. 59
      Murthy, S.; Reddy, G. P. J. Cell. Physiol. 2006, 209, 711– 717 DOI: 10.1002/jcp.20842
      59
      Replitase: complete machinery for DNA synthesis
      Murthy, Shalini; Reddy, G. Prem-Veer
      Journal of Cellular Physiology (2006), 209 (3), 711-717CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Liss, Inc.)
      A review. Replication of nuclear DNA in eukaryotes presents a tremendous challenge, not only due to the size and complexity of the genome, but also because of the time constraint imposed by a limited duration of S-phase during which the entire genome has to be duplicated accurately and only once per cell division cycle. A challenge of this magnitude can only be met by the close coupling of DNA precursor synthesis to replication. Prokaryotic systems provide evidence for multienzyme and multiprotein complexes involved in DNA precursor synthesis and DNA replication. In addn., fractionation of nuclear proteins from proliferating mammalian cells shows co-sedimentation of enzymes involved in DNA replication with those required for synthesis of deoxyribonucleoside triphosphates (dNTPs). Such complexes can be isolated only from cells that are in the S-phase, but not from cells in the G0/G1-phases of the cell cycle. The kinetics of deoxyribonucleotide metab. supporting DNA replication in intact and permeabilized cells reveals close coupling and allosteric interaction between the enzymes of dNTP synthesis and DNA replication. These interactions contribute to channeling and compartmentation of deoxyribonucleotides in the microvicinity of DNA replication. A multienzyme and multiprotein megacomplex with these unique properties is called "replitase.". Here, the authors summarize some of the relevant evidence to date that supports the concept of replitase in mammalian cells, which originated from observations in the lab. of A. B. Pardee (G. P. Reddy and A. B. Pardee, 1980.). In addn., the authors show that androgen receptor (AR), which plays a crit. role in proliferation and viability of prostate cancer cells, is assocd. with replitase, and that identification of the constituents of replitase in androgen-dependent vs. androgen-independent prostate cancer cells may provide insights into androgen-regulated events that control proliferation of prostate cancer cells and potentially offer an effective strategy for the treatment of prostate cancer.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtFOktLrI&md5=22356e76992b640ee798bec79c7e7aad
    60. 60
      Mathews, C. K. In Genetic Consequences of Nucleotide Pool Imbalance; de Serres, F. J., Ed.; Springer: Boston, MA, 1985.
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b11404.

    • Scheme S1, Methods, Supporting Figures S1–S6, Supporting Tables S1 and S2, Supporting References (PDF)


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

back