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Using Fitness Landscapes to assess RNA Structure-Function Relationships From Inside the Cell

使用适应度景观评估细胞内部的 RNA 结构-功能关系

基本信息

批准号：
10615093
负责人：
Michelle Margaret Meyer
金额：
$ 39.13万
依托单位：
BOSTON COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10615093
关键词：
Address Antibiotics Bacillus subtilis Bacterial RNA Behavior Binding Biological Biological Assay Biological Models Biological Process Cells Cellular Structures Characteristics Collection Complex Consumption Coupled Data Data Analyses Encapsulated Environment Flavin Mononucleotide Fluorescence Gene Expression Gene Expression Profile Gene Expression Profiling Gene Expression Regulation Glean Glycine Growth High-Throughput Nucleotide Sequencing In Vitro Individual Infection Life Ligand Binding Literature Maps Measurement Measures Mediating Methods Microfluidics Modeling Mus Mutation Nature Nucleotides Outcome Pharmacologic Substance Phenotype Play Point Mutation Population Positioning Attribute Public Health RNA RNA Folding RNA Sequences Reporter Research Ribosomes Role Small RNA Sorting Spliceosomes Streptococcus pneumoniae Structure Structure-Activity Relationship Study models Techniques Time Translating Variant Work antimicrobial antitumor agent aptamer bacterial fitness biophysical analysis biophysical model design experimental study fitness in vitro Assay innovation mouse model mutant novel prevent protein complex reconstitution response small molecule therapeutic target tool transcription termination

项目摘要

Summary RNA is an important therapeutic target, yet most RNA sequence-structure-function analysis is conducted in vitro rather than in the context of the cell despite substantive differences between the two environments. The objective of this proposal is to explore RNA sequence/function relationships within the cell using local fitness landscapes. Our model systems for this work are bacterial RNA regulators, such as riboswitches, which are antimicrobial targets and important biophysical models for studying RNA sequence-function relationships. Based on previous studies in the literature and our own preliminary data, we hypothesize that in vitro experiments imperfectly capture the sequence requirements required for biological function for many structured RNAs. To address how in vitro measured parameters correspond with cellular expression and organismal fitness, we plan to use three bacterial RNA regulators as model systems: the glycine riboswitch in Bacillus subtilis, the pyrR regulator in Streptococcus pneumoniae, and an FMN riboswitch in S. pneumoniae. Our specific aims are: 1) Determine which sequence changes to an RNA regulator impact organismal fitness and how these may be altered under different conditions that include defined culture medium, the presence of antibiotics, and a mouse infection model. 2) Establish the relationship between cellular gene expression profile and organismal phenotype. 3) Assess how gene expression and organismal fitness correspond with in vitro structure and function assays. We will achieve these aims by creating local fitness landscapes for each RNA regulator wherein all single and most double mutants are created as a pool, and subsequently evaluated using high-throughput sequencing as a final readout. The pools of variants will be appraised using a variety of techniques including: organismal competitions in culture (B. subtilis and S. pneumoniae), in the presence of targeting antibiotics (S. pneumoniae), and in a mouse infection model (S. pneumoniae); as well as microfluidic encapsulation of microcolonies to enable FADS-based assessment of cellular gene expression and in vitro transcription termination parameters. These high-throughput observations will be followed by assessment of a subset of mutants to confirm the observed phenotypes and examine secondary structure both in vitro and within the cell (SHAPE-MaP). Our proposal leverages organismal phenotypes we discovered in previous work and is an innovative approach to assess RNA sequence-function relationships that cannot be assessed in any other way. The expected outcome of the proposed work is the first mapping between RNA regulator sequence and a range of variables including organismal phenotype, gene expression, and in vitro termination characteristics. This contribution is significant because it will enable better interpretation of results from traditional in vitro and gene expression assays to inform efforts to target RNA regulators with antibiotics and to design novel RNA regulators.

总结 RNA是一个重要的治疗靶点，但大多数RNA序列-结构-功能分析是在在体外而不是在细胞环境中，尽管两种环境之间存在实质性差异。的这个建议的目的是探索RNA序列/功能的关系，在细胞内使用本地健身的风景.我们这项工作的模型系统是细菌RNA调节器，如核糖开关，抗菌目标和重要的生物物理模型研究RNA序列功能的关系。基于以前的研究文献和我们自己的初步数据，我们假设，在体外实验不完全捕获生物功能所需的序列要求， RNA。为了解决体外测量的参数如何与细胞表达和生物体表达相对应，为了适应性，我们计划使用三种细菌RNA调节器作为模型系统：芽孢杆菌中的甘氨酸核糖开关，枯草芽孢杆菌中的pyrR调节子，肺炎链球菌中的pyrR调节子，以及S.肺炎。我们具体目标是：1）确定RNA调节子的哪些序列变化影响生物体适应性，在不同的条件下，包括确定的培养基，抗生素和小鼠感染模型。2)建立细胞基因表达谱之间的关系和生物体表型。3)评估基因表达和生物体适应性如何与体外结构和功能测定。我们将通过为每个RNA创建局部适应度景观来实现这些目标调节子，其中所有单突变体和大多数双突变体作为库产生，随后使用高通量测序作为最终读数。将使用各种方法对变体池进行评估，技术包括：文化中的生物竞争（B. subtilis和S.肺炎），在存在靶向抗生素（S. pneumoniae）和小鼠感染模型（S.肺炎）;以及微流体包封小菌落，以实现基于FADS的细胞基因表达评估，转录终止参数。这些高通量观察之后将评估突变体的子集，以确认观察到的表型，并检查体外和在细胞内（SHAPE-MAP）。我们的建议利用了我们在以前的工作中发现的生物表型是一种评估RNA序列-功能关系的创新方法，别的办法了这项工作的预期成果是首次绘制出RNA调节子序列之间的图谱，以及一系列变量，包括生物体表型、基因表达和体外终止特色这一贡献是重要的，因为它将有助于更好地解释结果，传统的体外和基因表达测定，为用抗生素靶向RNA调节剂的努力提供信息，设计新的RNA调节子。