Accelerated evolution of biological RNAs

生物RNA的加速进化

基本信息

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
项目类别：
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

项目摘要

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.

项目概要： RNA 具有多种生物学功能，因为它能够采用明确的结构。可以作为遗传物质，可以通过蛋白质聚合酶合成和扩增。在实验室中对不断进化的 RNA 群体施加选择压力能够发现具有用户指定特性的功能性RNA，这一过程，即RNA的定向进化，已被广泛应用。用于生成基于 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的许多研究项目都有帮助，但目前它们的开发模式效率低下，限制了它们的效用。用于进化RNA的错误倾向不足以驱动功能基序的快速进化通常包含 20-50 个核苷酸。拟议的研究将开发一种容易出错的序列。具有强大 RNA 聚合酶活性的无偏 RNA 酶，专为加速定向进化而定制在目标 1 中，一种容易出错的聚合酶核酶，能够合成多种RNA。功能性 RNA 将适应诱变 RNA 群体的规模化生产，用于为了实现这一目标，一种新型的基于微流体的 RNA 进化平台已经问世。已经设计出来，并且其在当前形式的 RNA 聚合酶核酶中的应用已经得到验证。同时，在目标2中，将优化聚合反应条件以实现所需的每个核苷酸位置的错误率在 2-5% 范围内，具有广泛且无偏见的突变谱，从而实现功能性 RNA 基序的高效诱变，成为加速 RNA 进化的工具。目标 1 和 2 中开发的成果将应用于目标 3，以生成新的和改进的芒果和凝血因子 IXa 适体，分别可用于细胞成像和抗凝。这里开发的加速RNA进化平台将广泛增强生成生物界面的能力分子探针和适体是 RNA 定向进化的先驱。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 的选择 RNA、进化群体的深度测序和生物信息学分析，以及细胞成像索尔克研究所和更广泛的拉霍亚研究界已经改进了荧光适体。在基础和应用生物医学研究方面具有卓越且多样化的能力，这将为 PI 提供为成为一名成功的独立调查员提供高度培育的环境。