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.

项目摘要： RNA基于其采用明确定义的结构的能力，能够产生潜水员的生物学功能。 RNA也是可以用作可以通过蛋白质聚合酶合成和扩增的遗传物质。这在实验室中，在不断发展的RNA人群中施加选择性压力可以发现具有用户指定属性的功能性RNA。这个过程是RNA的定向演变，已广泛采用用于生成基于RNA的诊断，治疗，细胞调节元件和生物成像工具。这种合成功能RNA现在对许多生物学和生物医学研究计划有帮助，但是目前的开发范式效率低下，并限制了效用。蛋白质聚合酶用于进化RNA不足以易于错误，可以驱动功能基序的快速演变通常包含20-50个核动肽。拟议的研究将开发出易错的和序列 - 具有鲁棒RNA聚合酶活性的无偏RNA酶，量身定制，以加速定向演化功能性RNA。在AIM 1中，能够合成多样化的易易受错误的聚合酶核酶功能性RNA将适应RNA的诱变种群的缩放生产指导进化运动。为了实现这一目标，一个新型的基于微流体的RNA Evolution平台具有已设计并应用于RNA聚合酶核酶的当前形式。同时，在AIM 2中，将优化聚合反应的条件以实现所需每个核苷酸位置2-5％的错误率，突变的广泛频谱，从而实现了功能性RNA基序的产物诱变。加速RNA演化的工具然后将在目标1和2中开发凝血因子IXA适体，分别在细胞成像和抗凝作用中有用。此处开发的加速RNA进化平台将广泛增强产生生物互化的能力分子问题和适体。乔伊斯实验室开创了RNA的定向演变。这实验室的专业知识将有助于成功完成拟议的进化运动并促进了更广泛的科学界采用加速的RNA演化平台。 Salk Institute设有最先进的科学核心设施，这些设施将支持基于FACS的选择 RNA，深度测序和生物信息学分析的不断发展的种群以及细胞成像改善的荧光体能力。 Salk Institute和更广泛的La Jolla研究社区基本和应用生物医学研究的显着和潜水员的能力，该研究将为PI提供高度培养的环境成为成功的独立研究者。