Department of Chemistry and Center for Protein and Nucleic Acid Research, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
J Am Chem Soc. 2011 Dec 14;133(49):19878-88. doi: 10.1021/ja207907d. Epub 2011 Nov 18.
Site-specific labeling of enzymatically synthesized DNA or RNA has many potential uses in basic and applied research, ranging from facilitating biophysical studies to the in vitro evolution of functional nucleic acids and the construction of various nanomaterials and biosensors. As part of our efforts to expand the genetic alphabet, we have developed a class of unnatural base pairs, exemplified by d5SICS-dMMO2 and d5SICS-dNaM, which are efficiently replicated and transcribed, and which may be ideal for the site-specific labeling of DNA and RNA. Here, we report the synthesis and analysis of the ribo- and deoxyribo-variants, (d)5SICS and (d)MMO2, modified with free or protected propargylamine linkers that allow for the site-specific modification of DNA or RNA during or after enzymatic synthesis. We also synthesized and evaluated the α-phosphorothioate variant of d5SICSTP, which provides a route to backbone thiolation and an additional strategy for the postamplification site-specific labeling of DNA. The deoxynucleotides were characterized via steady-state kinetics and PCR, while the ribonucleosides were characterized by the transcription of both a short, model RNA as well as full length tRNA. The data reveal that while there are interesting nucleotide and polymerase-specific sensitivities to linker attachment, both (d)MMO2 and (d)5SICS may be used to produce DNA or RNA site-specifically modified with multiple, different functional groups with sufficient efficiency and fidelity for practical applications.
酶促合成的 DNA 或 RNA 的位点特异性标记在基础和应用研究中有许多潜在用途，从促进生物物理研究到功能核酸的体外进化以及各种纳米材料和生物传感器的构建。作为我们扩展遗传密码子努力的一部分，我们开发了一类非天然碱基对，以 d5SICS-dMMO2 和 d5SICS-dNaM 为代表，它们能够有效地复制和转录，并且可能非常适合 DNA 和 RNA 的位点特异性标记。在这里，我们报告了核糖和脱氧核糖变体 (d)5SICS 和 (d)MMO2 的合成和分析，它们经过修饰后带有游离或保护的炔丙胺连接子，允许在酶促合成过程中或之后对 DNA 或 RNA 进行位点特异性修饰。我们还合成并评估了 d5SICSTP 的 α-硫代磷酸酯变体，它为骨架硫代化提供了一种途径，并为 DNA 的扩增后位点特异性标记提供了另一种策略。通过稳态动力学和 PCR 对脱氧核苷酸进行了表征，而通过对短模型 RNA 和全长 tRNA 的转录对核糖核苷进行了表征。数据表明，尽管存在有趣的核苷酸和聚合酶特异性对连接子附着的敏感性，但 (d)MMO2 和 (d)5SICS 都可用于以足够的效率和保真度产生 DNA 或 RNA 的位点特异性修饰，带有多个不同的功能基团，可用于实际应用。
