RNA Folding and Adaptation in a Cellular Context

细胞环境中的 RNA 折叠和适应

• 批准号: 8901235
• 负责人: PHILIP C BEVILACQUA
• 金额: $ 27.37万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8901235
• 关键词: Address; Base Pairing; Behavior; Binding; Biological; Biological Phenomena; Biological Process; Biophysics; Biopolymers; Calorimetry; Catalytic RNA; Cells; Chemicals; Collaborations; Cytoplasm; DNA; Data; Engineering; Environment; Enzymes; Eukaryotic Cell; Genetic Programming; Genetic Transcription; Genotype; Goals; Guide RNA; Health; Human; Human Genome; In Vitro; Kinetics; Laboratories; Lead; Length; Libraries; Life; Maps; Measures; Methods; MicroRNAs; Modeling; Molecular; Mutation; Nature; Outcome; Phase; Phenotype; Physiological; Pilot Projects; Polymers; Process; Prokaryotic Cells; Proteins; RNA; RNA Folding; RNA library; Relative (related person); Research; Role; Series; Shapes; Small Interfering RNA; Structure; Tertiary Protein Structure; Testing; Theoretical Studies; Theoretical model; Therapeutic; Thermodynamics; Untranslated RNA; Variant; Virus; aptamer; base; biophysical properties; cellular imaging; design; driving force; experience; in vivo; insight; interest; member; next generation sequencing; novel; novel strategies; pressure; research study; temperature jump; therapeutic development

DESCRIPTION (provided by applicant): Folding of RNA in the cell is not well understood nor has it been integrated into a cohesive mechanistic framework. The broad objectives of this proposal are to develop comprehensive molecular mechanisms for how functional RNAs fold in vivo and to relate these mechanisms to the evolutionary forces that help shape them. A comprehensive approach will be taken in which both the biophysical and evolutionary driving forces that give rise to RNA folding mechanism in vivo will be identified. The first specific aim will establish biophysical principles for in vivo RNA folding by examining the folding mechanisms of several naturally occurring riboswitches and ribozymes in both model cytoplasms and in cells. The second specific aim will elucidate evolutionary principles that guide RNA folding in vivo by testing the folding mechanisms of sequences that will emerge from several neutral drift selections. Thus, both naturally occurring and laboratory-evolved functional RNAs will be examined, with an overall goal of elucidating general principles for RNA folding in the cell. The research involves developing a series of model cytoplasms and testing the folding cooperativity and kinetics of RNAs in these. In addition, studies of folding mechanism will be conducted directly in eukaryotic and prokaryotic cells using several novel approaches. The role of evolutionary forces in shaping RNA folding landscapes in vivo will be studied by conducting several neutral drift selections. Members of these libraries will be assessed for folding thermodynamics and kinetics. The ability of cooperatively folding RNAs to adapt to selective pressures will also be assessed. Methods to be applied throughout this research include high-throughput calorimetry; CD and UV-detected thermal denaturation; rapid kinetics; SAXS; and the expression, structure mapping, and live cell imaging of RNAs in various cells. Data throughout the research will be modeled by several theoretical and computational approaches, which will be used both to help understand folding behavior and to refine experiments. Because new insights into RNA folding dynamics and adaptation should be revealed, the results should broadly influence many different health- related projects. The findings may make it possible to rationally engineer RNA therapeutics with different in vivo stabilities, and they may lead to new insights into the relationship between genotype and phenotype in viruses.

描述（由申请人提供）：细胞中 RNA 的折叠尚未被充分理解，也没有被整合到一个内聚的机制框架中。该提案的主要目标是开发功能性 RNA 在体内如何折叠的全面分子机制，并将这些机制与帮助塑造它们的进化力量联系起来。将采取综合方法，确定体内RNA折叠机制的生物物理和进化驱动力。第一个具体目标是通过检查模型细胞质和细胞中几种天然存在的核糖开关和核酶的折叠机制，建立体内 RNA 折叠的生物物理学原理。第二个具体目标是通过测试从几个中性漂移选择中出现的序列的折叠机制来阐明指导体内RNA折叠的进化原理。因此，天然存在的和实验室进化的功能性RNA都将被检查，总体目标是阐明细胞中RNA折叠的一般原理。该研究涉及开发一系列细胞质模型并测试这些细胞质中 RNA 的折叠协同性和动力学。此外，折叠机制的研究将使用几种新方法直接在真核和原核细胞中进行。将通过进行几次中性漂移选择来研究进化力量在塑造体内 RNA 折叠景观中的作用。这些库的成员将接受折叠热力学和动力学评估。合作折叠 RNA 适应选择压力的能力也将被评估。本研究中应用的方法包括高通量量热法； CD 和 UV 检测的热变性；快速动力学；萨克斯；以及各种细胞中 RNA 的表达、结构图谱和活细胞成像。整个研究中的数据将通过多种理论和计算方法进行建模，这些方法将用于帮助理解折叠行为并完善实验。由于应该揭示对 RNA 折叠动力学和适应的新见解，因此结果应该广泛影响许多不同的健康相关项目。这些发现可能使得合理设计具有不同体内稳定性的RNA疗法成为可能，并且可能带来对病毒基因型和表型之间关系的新见解。