Recognition of Synthetic Unnatural Base Pairs by RNA Polymerase

RNA 聚合酶对合成非天然碱基对的识别

• 批准号: 10561543
• 负责人: Dong Wang
• 金额: $ 40.33万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-15 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10561543
• 关键词: Active Sites; Adenine; Amino Acids; Bacteriophage T7; Base Pairing; Binding; Biochemical; Biochemistry; Biology; Biophysics; Chemicals; Chemistry; Complex; Computational Biology; Cryoelectron Microscopy; Cytosine; DNA; DNA-Directed RNA Polymerase; Data; Development; Diagnosis; Enzymes; Escherichia coli; Eukaryota; Family; Foundations; Future; Genetic; Genetic Transcription; Goals; Guanine; Hydrogen Bonding; Hydrophobicity; Information Storage; Kinetics; Knowledge; Label; Life; Methods; Mission; Molecular; Nucleic Acids; Nucleotides; Organism; Outcome; Planet Earth; Polymerase; Prokaryotic Cells; Proteins; Public Health; RNA; RNA Polymerase II; Reaction; Research; Retrieval; Series; Shapes; Structure; T7 RNA polymerase; Testing; Thymine; United States National Institutes of Health; Uracil; X-Ray Crystallography; Yeasts; design; functional group; genetic information; human disease; in vivo; insight; next generation; novel; novel therapeutics; nucleic acid-based therapeutics; structural biology; synthetic biology; synthetic nucleotide; therapeutic protein; tool

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.

项目概要/摘要 地球上的所有生命形式都使用同一组自然遗传字母：腺嘌呤 (A)、胞嘧啶 (C)、鸟嘌呤 (G)、胸腺嘧啶 (T)（和尿嘧啶 (U)）作为存储和检索其遗传信息的构建模块。 最近，在开发第一个能够存储的半合成生物体方面取得了重大突破 并检索体内含有非自然碱基对（UBP）的遗传信息。然而，分子基础 UBP 的转录加工过程尚不清楚。一个重要且长期悬而未决的问题仍然存在 未解答：这些 UBP 是如何被细胞转录机制识别的？缺乏明确的答案 这个重要问题代表了该领域的主要知识差距。该项目的长期目标是 解决这个重要问题。我们假设 UBP 的转录识别由两层控制 特异性相互作用：非天然核酸模板和底物之间的特异性相互作用 因为它们与 RNA 聚合酶活性位点的相互作用。我们将进行动力学研究并比较 不同RNA聚合酶对三类代表性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制见解 核苷酸底物并催化核苷酸加成反应。我们将采用一种组合方法 包括 X 射线晶体学、冷冻电镜、生物物理学、生物化学、计算生物学和核酸 化学。所提出的研究具有重大意义和开创性，因为新颖的知识和 从这项拟议研究中获得的结构将对转录领域产生变革性影响， 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 核酸相互作用以及非天然核酸和核苷酸如何被不同的 RNA 识别 聚合酶。最终，这些知识将为开发下一代 UBP 和 将产生含有新功能基团的新型治疗性核酸和蛋白质。