Generation of Chimeric DNA-RNA Structures using Engineered Replisomes in vivo

使用体内工程复制体生成嵌合 DNA-RNA 结构

• 批准号: 10227556
• 负责人: Braddock Sandoval
• 金额: $ 6.56万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227556
• 关键词: Active Sites; Alkynes; Amino Acids; Bacteriophage T7; Biochemical; Biological; Biological Assay; Biology; Biopolymers; Biotechnology; Cells; Chemical Structure; Chemicals; Chimera organism; Complement; Complex; DNA; DNA biosynthesis; DNA-Directed DNA Polymerase; Deoxyribose; Deoxyuridine; Development; Engineering; Enzymes; Escherichia coli; Generations; Genes; Genetic Structures; Genome; Genomic DNA; Goals; In Vitro; Information Storage; Label; Libraries; Life; Link; Methods; Modernization; Monitor; Mutagenesis; Mutation; Nucleotides; Phage Display; Plasmids; Polymerase; Procedures; Protein Engineering; Protocols documentation; RNA; Reaction; Reading; Research; Retrieval; Ribonucleotides; Ribose; Ribosomes; Role; Science; Specificity; Structure; Structure-Activity Relationship; Substrate Specificity; System; Technology; Time; Uridine; Viral Genome; analog; base; cofactor; experimental study; fascinate; genetic information; in vivo; monomer; mutant; novel; novel strategies; polymerization; rapid technique; screening; sugar; synthetic biology; tool; tripolyphosphate

PROJECT SUMMARY/ABSTRACT The evidence of ribose in ancient catalytic machinery such as the ribosome as well as a universal set of cofactors which are derived from ribonucleic acids (RNA) has led to the hypothesis that RNA was once the dominant biopolymer in life. The transition from an RNA to DNA world raises a multitude of questions regarding how life was able to support such a dramatic change in the most fundamental information storage molecules without compromising survival. Recent studies have shown that certain randomly mutagenized strains of E. coli are in fact capable of supporting surprisingly high amounts of RNA in their genome, however it remains unclear the exact biochemical mechanisms that allow this to occur, and further studies of these strains is challenging due to inherent instability of their genomes. In this project I will be developing a novel polymerase system which could potentially limit RNA incorporation to small regions of DNA such as a plasmid. Targeting RNA incorporation in this way will allow for the first time a systematic method to study the effects of high ribose content in genes, without compromising host genomes. The proposal outlined here will focus on strategies for engineering the DNA polymerase within the multienzyme viral genome replication machinery known as the T7 replisome. In Specific Aim I, I will outline a plan to study the effects of focused mutations within the active site of T7 DNA polymerase. This will include the development and implementation of 96-well based screening method for the rapid determination of DNA polymerases with the ability to incorporate RNA permissively. Specific Aim II details a protocol for the selection of RNA permissive DNA polymerases from much larger mutant pools. A key feature of this aim is the combination of phage-display which has been used for the selection of nucleotide promiscuity in vitro along with a complementation-based selection strategy which further selects for mutant polymerases that retain functionality in vivo. We hypothesize that a novel combination of these strategies can be used to solve the inherent limitation of phage display to in vitro reaction conditions which may not accurately reflect intracellular conditions. Finally, Specific Aim III details a new approach toward the selection of mutant polymerases with the ability to incorporate RNA into replicating plasmids within a bacterial host. This method will rely on the use of alkyne labeled ribonucleotides which will provide chemical handles for the enrichment of plasmids that encode polymerases with relaxed sugar specificity. Together this project will create progress toward an orthogonal polymerase system for the construction of chimeric DNA-RNA plasmids in vivo, a task which is currently impossible using known synthetic biology tools.

项目摘要/摘要 古代催化机械中核糖的证据，例如核糖体以及一套通用的辅助因子 源自核糖核酸（RNA）的，这导致了以下假设：RNA曾经是主要的 生命中的生物聚合物。从RNA到DNA世界的过渡提出了许多有关生活的问题 能够支持最基本的信息存储分子的这种巨大变化 损害生存。最近的研究表明，大肠杆菌的某些随机诱变菌株 能够支持其基因组中令人惊讶的大量RNA的事实，但是尚不清楚 确切的生化机制可以实现这一目标，并且由于 其基因组的固有不稳定。在这个项目中，我将开发一个新型的聚合酶系统，可以 潜在地将RNA掺入到DNA的小区域，例如质粒。靶向RNA掺入 这种方式将首次允许一种系统的方法来研究基因中高核糖含量的影响， 没有损害宿主基因组。 此处概述的提案将重点介绍多烯酶内部设计DNA聚合酶的策略 病毒基因组复制机制称为T7重建体。在特定目标中，我将概述研究 T7 DNA聚合酶活性位点中聚焦突变的影响。这将包括开发以及 实施96孔的筛选方法，以快速测定DNA聚合酶 能够允许地合并RNA。特定目标II详细介绍了选择RNA允许性的方案 来自更大突变池的DNA聚合酶。这个目标的关键特征是噬菌体播放的组合 它已用于在体外选择核苷酸杂交以及基于互补的 选择策略进一步选择了在体内保留功能的突变聚合酶。我们假设 这些策略的新型组合可以用来解决噬菌体显示的固有限制到 体外反应条件可能无法准确反映细胞内条件。最后，特定的目标III细节 一种新的方法，用于选择突变聚合酶，并能够将RNA纳入复制 细菌宿主中的质粒。此方法将依靠使用炔烃标记为核糖核苷酸的使用 提供化学手柄，以富集具有松弛糖特​​异性的聚合酶的质粒。 这个项目将共同朝着正交聚合酶系统建设的进展 体内嵌合DNA-RNA质粒，这项任务目前使用已知的合成生物学工具是不可能的。