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: 10474282
• 负责人: Braddock Sandoval
• 金额: $ 6.72万
• 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/10474282
• 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.

项目总结/摘要 核糖存在于古老的催化机制中的证据，如核糖体以及一组通用的辅因子 来自核糖核酸（RNA）的DNA的研究导致了这样的假设，即RNA曾经是占主导地位的 生命中的生物聚合物从RNA世界到DNA世界的转变引发了许多关于生命如何生存的问题 能够在最基本的信息存储分子中支持如此巨大的变化， 危及生存最近的研究表明，某些随机诱变的E。大肠杆菌在 事实上，能够支持其基因组中令人惊讶的大量RNA，但目前仍不清楚 确切的生化机制，使这种情况发生，这些菌株的进一步研究是具有挑战性的， 其基因组固有的不稳定性。在这个项目中，我将开发一种新的聚合酶系统， 潜在地将RNA掺入限制在DNA的小区域，例如质粒。靶向RNA掺入 这种方法将首次允许系统的方法来研究基因中高核糖含量的影响， 而不损害宿主基因组 这里概述的建议将集中在多酶中DNA聚合酶的工程策略上。 病毒基因组复制机制称为T7复制体。在具体目标I中，我将概述一个研究 T7 DNA聚合酶活性位点内集中突变的影响。这将包括发展和 用96孔板筛选法快速测定DNA聚合酶 允许掺入RNA的能力。Specific Aim II详细介绍了选择RNA允许的 DNA聚合酶从更大的突变池。这一目标的一个关键特征是噬菌体展示与细胞外基质的结合。 其已用于体外选择核苷酸混杂性，沿着基于互补的 选择策略，其进一步选择在体内保留功能性的突变聚合酶。我们假设 这些策略的新组合可用于解决噬菌体展示的固有局限性， 可能不能准确反映细胞内条件的体外反应条件。第三章具体目标 一种新的选择突变聚合酶的方法， 细菌宿主中的质粒。该方法将依赖于使用炔标记的核糖核苷酸，其将 提供用于富集编码具有松弛糖特异性的聚合酶的质粒的化学手柄。 该项目将共同创造一个正交聚合酶系统的建设进展， 体内嵌合DNA-RNA质粒，这是目前使用已知合成生物学工具不可能完成的任务。