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

基本信息

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
项目类别：
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

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

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项目摘要

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.

项目摘要/摘要地球上的所有生命形式都使用相同的自然遗传字母：腺嘌呤（a），胞嘧啶（C），鸟嘌呤（g），胸腺嘧啶（t）（和乌拉西尔（U））作为存储和检索其遗传信息的基础。最近，在开发第一个能够存储的第一个半合成有机体中取得了重大突破并在体内检索包含不自然基对（UBP）的遗传信息。但是，分子基础 UBP的转录处理的理解很少。仍然存在一个重要且长期存在的问题未解决：这些UBP如何通过细胞转录机械识别？缺乏明确的答案这个重要的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作为与RNA聚合酶活性位点的相互作用。我们将进行动力学研究，并比较通过不同的RNA聚合酶对三类代表性UBP的转录处理，包括单盐和多亚基RNA聚合酶。我们将确定转录的结构基础识别UBPS并获得有关转录机器如何识别不自然的机械见解核苷酸底物和催化核苷酸添加反应。我们将利用一种合并的方法包括X射线晶体学，冷冻，生物物理学，生物化学，计算生物学和核酸化学。拟议的研究是重要的，而且是突破性的，因为新颖的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变革性的影响，核酸化学以及合成生物学，并垂直提高我们对蛋白质的理解核酸相互作用以及如何通过不同的RNA识别不自然的核酸和核苷酸聚合酶。最终，此类知识将为开发下一代UBP和将产生新型的治疗核酸和含有新功能组的蛋白质。