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

基本信息

批准号：
10700911
负责人：
Braddock Sandoval
金额：
$ 6.95万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
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/10700911
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 Mutagens Mutation Nucleotides Phage Display Plasmids Polymerase Polymers Procedures Protein Engineering Protocols documentation RNA Reaction Relaxation Research Retrieval Ribonucleotides Ribose Ribosomes Role Science Sorting Specificity Structure Structure-Activity Relationship Substrate Specificity System Technology Time Uridine Viral Genome Virus Replication analog cofactor experimental study fascinate genetic information in vivo monomer mutant novel novel strategies permissiveness polymerization rapid technique screening sugar synthetic biology tool tripolyphosphate

项目摘要

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.

项目概要/摘要古代催化机制（如核糖体）中存在核糖的证据以及一组通用的辅助因子衍生自核糖核酸 (RNA) 的物质导致了这样的假设：RNA 曾经是主要的生活中的生物聚合物。从 RNA 世界到 DNA 世界的转变引发了许多关于生命如何存在的问题能够支持最基本的信息存储分子的如此巨大的变化，而无需危及生存。最近的研究表明，某些随机诱变的大肠杆菌菌株存在于事实上，它们的基因组中能够支持令人惊讶的大量 RNA，但目前尚不清楚导致这种情况发生的确切生化机制，并且由于以下原因对这些菌株的进一步研究具有挑战性他们的基因组固有的不稳定性。在这个项目中，我将开发一种新型聚合酶系统，该系统可以潜在地限制 RNA 掺入 DNA 的小区域，例如质粒。靶向 RNA 掺入这种方法将首次提供一种系统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。这里概述的提案将重点关注在多酶中设计 DNA 聚合酶的策略病毒基因组复制机制称为 T7 复制体。在具体目标 I 中，我将概述一个研究计划 T7 DNA 聚合酶活性位点内集中突变的影响。这将包括开发和实施基于 96 孔的快速测定 DNA 聚合酶的筛选方法允许掺入 RNA 的能力。具体目标 II 详细介绍了选择 RNA 许可的协议来自更大突变体库的 DNA 聚合酶。该目标的一个关键特征是噬菌体展示的结合它已被用于体外选择核苷酸混杂性以及基于互补的选择策略，进一步选择保留体内功能的突变聚合酶。我们假设这些策略的新颖组合可用于解决噬菌体展示的固有局限性体外反应条件可能无法准确反映细胞内条件。最后，具体目标 III 细节一种选择能够将RNA整合到复制中的突变聚合酶的新方法细菌宿主内的质粒。该方法将依赖于炔标记的核糖核苷酸的使用，该核糖核苷酸将为富集编码具有宽松糖特异性的聚合酶的质粒提供化学手柄。该项目将共同为构建正交聚合酶系统取得进展体内嵌合DNA-RNA质粒，这是目前使用已知的合成生物学工具不可能完成的任务。