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

项目摘要/摘要 在古老的催化机械中存在核糖的证据，如核糖体以及一组通用的辅因子 它们是由核糖核酸(Rna)衍生的，这导致了一种假设，即rna曾经是主要的 生命中的生物聚合物。从RNA世界到DNA世界的转变引发了一系列关于生命如何 能够支持最基本的信息存储分子的如此戏剧性的变化 危及生存。最近的研究表明，某些随机诱变的大肠杆菌菌株在 能够在它们的基因组中支持惊人数量的RNA的事实，然而，目前尚不清楚 导致这种情况发生的确切生化机制，以及对这些菌株的进一步研究是具有挑战性的，因为 它们基因组的内在不稳定性。在这个项目中，我将开发一种新型的聚合酶系统，它可以 潜在地将RNA掺入限制在DNA的小区域，如质粒。靶向RNA掺入 这种方法将首次允许系统地研究基因中高核糖含量的影响， 而不会损害宿主基因组。 这里概述的提案将集中于在多酶中设计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，我将概述一项研究 T7DNA聚合酶活性部位集中突变的影响。这将包括开发和 应用96孔筛选法快速测定DNA聚合酶 允许整合RNA的能力。特殊目的II详细介绍了一种选择RNA允许剂的方案 来自更大的突变库的DNA聚合酶。这一目标的一个关键特征是噬菌体展示的组合 它已被用于体外核苷酸混杂的选择以及基于互补的 进一步选择在体内保留功能的突变聚合酶的选择策略。我们假设 这些策略的一种新的组合可以用来解决噬菌体展示在 体外反应条件可能不能准确反映细胞内的条件。最后，具体目标三的细节 选择能够将RNA整合到复制中的突变型聚合酶的新方法 细菌宿主内的质粒。这种方法将依赖于使用炔基标记的核糖核苷酸，这将 为编码具有松弛糖专一性的聚合酶的质粒的浓缩提供化学处理。 该项目将共同创造一个用于构建正交聚合酶系统的进展 体内的嵌合DNA-RNA质粒，这是一项目前使用已知的合成生物学工具无法完成的任务。

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.