Accelerated evolution of biological RNAs

项目概要： RNA能够基于其采用明确定义的结构的能力而具有多种生物学功能。RNA也 可作为可通过蛋白质聚合酶合成和扩增的遗传物质。的 在实验室中对进化中的RNA群体施加选择压力， 具有用户指定属性的功能性RNA。这个过程，RNA的定向进化，已经被广泛地研究。 用于产生基于RNA的诊断、治疗、细胞调控元件和生物成像工具。 这种合成的功能性RNA现在对生物学和生物医学中的许多研究项目都是有用的， 但目前的发展模式效率低下，限制了它们的效用。蛋白质聚合酶 用于进化的RNA不足以容易出错，以驱动功能基序的快速进化， 通常含有20-50个核苷酸。拟议的研究将开发一个容易出错的序列- 无偏见的RNA酶，具有强大的RNA聚合酶活性，专为加速定向进化而设计， 功能性RNA。在Aim 1中，一种能够合成多种 功能性RNA将适合于规模化生产诱变的RNA群体， 定向进化运动为了实现这一目标，一种新的基于微流体的RNA进化平台已经 的设计和它的应用，目前形式的RNA聚合酶核酶已得到验证。 同时，在目标2中，将优化聚合反应的条件以实现期望的聚合反应。 每个核苷酸位置的错误率在2-5%的范围内，具有广泛且无偏的突变谱， 从而能够对功能性RNA基序进行生产性诱变。加速RNA进化的工具 在目标1和2中开发的技术将用于目标3，以产生新的和改进的芒果形式， 凝血因子IXa适体，其分别用于细胞成像和抗凝。的 这里开发的加速RNA进化平台将广泛增强产生生物界面的能力， 分子探针和适体。乔伊斯实验室是RNA定向进化的先驱。的 实验室的专业知识将有助于成功完成拟议的进化活动 并促进更广泛的科学界采用加速RNA进化平台。 索尔克研究所拥有最先进的科学核心设施，将支持基于外地资产管制系统的选择， 进化群体的RNA、深度测序和生物信息学分析，以及 改进的荧光适体。索尔克研究所和更广泛的拉霍亚研究社区已经 在基础和应用生物医学研究方面的卓越和多样化的能力，这将为PI提供 高度培养的环境，成为一个成功的独立调查员。

Project Summary: RNA is capable of diverse biological functions based on its ability to adopt well-defined structures. RNA also can serve as a genetic material that can be synthesized and amplified by protein polymerase enzymes. The application of selective pressures to an evolving population of RNAs in the laboratory enables the discovery of functional RNAs with user-specified properties. This process, the directed evolution of RNA, has been widely adopted to generate RNA-based diagnostics, therapeutics, cellular regulatory elements, and bio-imaging tools. Such synthetic functional RNAs are now instrumental to many research programs in biology and biomedicine, but the current paradigm for their development is inefficient and limits their utility. The protein polymerases that are used to evolve RNAs are not sufficiently error-prone to drive the rapid evolution of functional motifs that typically contain 20–50 nucleotides. The proposed research will develop an error-prone and sequence- unbiased RNA enzyme with robust RNA polymerase activity, tailored to accelerate the directed evolution of functional RNAs. In Aim 1, an error-prone polymerase ribozyme that is capable of synthesizing diverse functional RNAs will be adapted to the scaled production of mutagenized populations of RNAs for use in directed evolution campaigns. To achieve this goal, a novel microfluidics-based RNA evolution platform has been devised and its application to the current form of the RNA polymerase ribozyme has been validated. Simultaneously, in Aim 2, the conditions of the polymerization reaction will be optimized to achieve the desired error rates in the range of 2–5% per nucleotide position, with a broad and unbiased spectrum of mutations, thus enabling the productive mutagenesis of functional RNA motifs. The tools for accelerated RNA evolution developed in Aims 1 and 2 will then be applied in Aim 3 to generate new and improved forms of the Mango and coagulation factor IXa aptamers, which have utility in cellular imaging and anticoagulation, respectively. The accelerated RNA evolution platform developed here will broadly enhance the ability to generate bio-interfacing molecular probes and aptamers. The Joyce laboratory has pioneered the directed evolution of RNA. The laboratory’s expertise will be instrumental in the successful completion of the proposed evolution campaigns and in fostering the adoption of the accelerated RNA evolution platform by the broader scientific community. The Salk Institute houses state-of-the-art scientific core facilities that will support the FACS-based selection of RNA, deep sequencing and bioinformatics analyses of the evolving populations, and cellular imaging of the improved fluorescent aptamers. The Salk Institute and the broader La Jolla research community have remarkable and diverse capabilities in basic and applied biomedical research, which will provide the PI with a highly nurturing environment toward becoming a successful independent investigator.