Accelerated evolution of biological RNAs

生物RNA的加速进化

• 批准号: 10666401
• 负责人: Wesley Garret Cochrane
• 金额: $ 7.18万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10666401
• 关键词: Acceleration; Adopted; Adoption; Affect; Anticoagulants; Anticoagulation; Binding; Binding Sites; Bioinformatics; Biological; Biological Process; Biology; Catalytic RNA; Clinical; Coagulation Process; Communities; Complex; Core Facility; DNA; DNA-Directed RNA Polymerase; Development; Diagnostic; Directed Molecular Evolution; Emulsions; Environment; Enzymes; Evolution; Factor IXa; Fluorescence; Fostering; Frequencies; Gene Expression; Gene Expression Regulation; Genetic Materials; Goals; Heparin; Human; Imaging Device; Individual; Laboratories; Mammalian Cell; Mango - dietary; Methods; MicroRNAs; Microfluidics; Molecular; Molecular Probes; Mutagenesis; Mutagens; Mutation; Nucleotides; Plasma; Play; Polymerase; Polymers; Population; Population Heterogeneity; Positioning Attribute; Procedures; Process; Production; Productivity; Property; Proteins; Protocols documentation; RNA; RNA Sequences; RNA chemical synthesis; RNA primers; Reaction; Regulatory Element; Research; Research Personnel; Role; Selection Criteria; Specific qualifier value; Structure; Synthetic Genes; System; Testing; Therapeutic; Thromboplastin; Time; Untranslated RNA; Variant; applied biomedical research; aptamer; base; bioimaging; cellular imaging; deep sequencing; functional improvement; immunogenic; improved; intermolecular interaction; live cell imaging; molecular diagnostics; nanomolar; novel; nucleobase; polymerization; pressure; programs; protein complex; screening; tool

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.

项目总结： 基于其采用明确结构的能力，RNA具有多种生物学功能。核糖核酸也 可以作为一种遗传物质，可以通过蛋白质聚合酶合成和放大。这个 在实验室中对进化中的RNA群体施加选择性压力使发现 具有用户指定属性的功能RNA。这一过程，即rna的定向进化，已经被广泛应用。 用于产生基于RNA的诊断、治疗、细胞调节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计划都是有用的， 但目前它们的发展模式效率低下，并限制了它们的实用性。蛋白质聚合酶 用于进化RNA的RNA不太容易出错，以驱动功能基序的快速进化，这些功能基序 通常含有20-50个核苷酸。拟议的研究将制定一个容易出错的顺序-- 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定制 功能核糖核酸。在目标1中，一种容易出错的聚合酶核酶能够合成不同的 功能性RNA将被改造成规模化生产突变的RNA种群，用于 指导进化运动。为了实现这一目标，一种新型的基于微流控的RNA进化平台 已经被设计出来，它在当前形式的RNA聚合酶核酶中的应用已经得到验证。 同时，在目标2中，聚合反应的条件将被优化以达到预期的效果 错误率在每个核苷酸位置2-5%的范围内，具有广泛和公正的突变谱， 从而使功能性RNA基序能够产生突变。加速RNA进化的工具 在目标1和目标2中开发的芒果将在目标3中应用，以生成新的和改进的芒果和 凝血因子IXa适配子，分别用于细胞成像和抗凝。这个 这里开发的加速RNA进化平台将广泛增强产生生物接口的能力 分子探针和适配子。乔伊斯实验室开创了RNA定向进化的先河。这个 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FACS的 RNA，进化种群的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界 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 为成为一名成功的独立调查员创造了良好的环境。