Recognition of Synthetic Unnatural Base Pairs by RNA Polymerase

项目摘要/摘要 地球上所有的生命形式都使用相同的一套自然遗传字母表：腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤 (G)、胸腺嘧啶(T)(和尿嘧啶(U))作为储存和检索其遗传信息的构件。 最近，在开发第一个能够储存的半合成生物方面取得了重大突破 并在体内检索包含非自然碱基对(UBP)的遗传信息。然而，分子基础 对UBP的转录过程的了解很少。一个重要而长期存在的问题仍然存在 未回答：这些UBP是如何被细胞转录机器识别的？缺乏明确的答案 这一重要问题代表了该领域的一个重大知识差距。这个项目的长期目标是 解决这个重要的问题。我们假设UBP的转录识别由两层控制 特定相互作用：非天然核酸模板与底物之间的特定相互作用 因为它们与RNA聚合酶的活性部位相互作用。我们将进行动力学研究，并比较 不同的RNA聚合酶对三类具有代表性的UBP的转录加工，包括 单亚基和多亚基RNA聚合酶。我们将确定转录的结构基础 识别UBP并获得转录机器如何识别非自然的机械性见解 核苷酸底物和催化核苷酸加成反应。我们将利用一种组合方法，即 包括X射线结晶学、低温电子显微镜、生物物理学、生物化学、计算生物学和核酸 化学反应。这项拟议的研究具有重大意义和开创性，因为新知识和 从这项拟议的研究中获得的结构将对转录领域产生革命性的影响， 核酸化学，以及合成生物学，并垂直推进我们对蛋白质的理解- 核酸的相互作用以及不同的RNA如何识别非自然的核酸和核苷酸 聚合酶。归根结底，这些知识将为开发下一代无人驾驶飞机和 将产生含有新功能基团的新型治疗性核酸和蛋白质。

Project Summary/Abstract All life forms on Earth use the same set of natural genetic alphabets: adenine (A), cytosine (C), guanine (G), thymine (T) (and uracil (U)) as the building blocks for storage and retrieval of their genetic information. Recently, a major breakthrough was made in developing the first semi-synthetic organism that is able to store and retrieve genetic information containing an unnatural base pair (UBP) in vivo. However, the molecular basis of transcription processing of UBPs is poorly understood. An important and long-standing question remains unanswered: How are these UBPs recognized by cellular transcription machinery? A lack of clear answers to this important question represents a major knowledge gap in the field. The long-term goal of this project is to tackle this important question. We hypothesize the transcription recognition of UBPs is governed by two layers of specific interactions: specific interactions between the unnatural nucleic acid template and substrates as well as their interplays with the active site of RNA polymerase. We will perform kinetic studies and compare the transcription processing of three classes of representative UBPs by different RNA polymerases, including single-subunit and multi-subunit RNA polymerases. We will determine the structural basis of transcription recognition of UBPs and gain the mechanistic insights into how transcription machineries recognize unnatural nucleotide substrate and catalyze the nucleotide addition reaction. We will utilize a combined approach that includes X-ray crystallography, cryoEM, biophysics, biochemistry, computational biology, and nucleic acid chemistry. The proposed research is significant and groundbreaking, because the novel knowledge and structures obtained from this proposed research will have a transformative impact on the fields of transcription, nucleic acid chemistry, as well as synthetic biology and vertically advance our understanding of the protein- nucleic acid interactions and how unnatural nucleic acids and nucleotides are recognized by different RNA polymerases. Ultimately, such knowledge will provide a framework for developing next generation of UBPs and would produce novel therapeutic nucleic acids and proteins containing new functional groups.