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的折叠尚未很好地理解，也没有将其整合到粘性机械框架中。该提案的广泛目标是开发全面的分子机制，以使功能性RNA在体内折叠并将这些机制与有助于塑造它们的进化力相关联。将采用一种全面的方法，即将确定在体内产生RNA折叠机制的生物物理和进化驱动力。第一个具体目的将通过检查模型细胞肿瘤和细胞中几种天然存在的核糖开关和核酶的折叠机制来建立生物物理原理，以建立体内RNA折叠的生物物理原理。第二个特定目的将通过测试将从几个中性漂移选择中出现的序列的折叠机制来指导体内引导RNA折叠的进化原理。因此，将检查自然发生的和实验室发展的功能RNA，其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模型的细胞肿块，并测试其中RNA的折叠合作性和动力学。此外，对折叠机制的研究将直接在真核和原核细胞中使用几种新方法进行。通过进行几种中性漂移选择，将研究进化力在体内塑造RNA折叠景观中的作用。这些图书馆的成员将用于折叠热力学和动力学。还将评估合作折叠RNA适应选择性压力的能力。整个研究中要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，结构映射和活细胞成像。整个研究过程中的数据将通过几种理论和计算方法进行建模，这些方法将既可以用来帮助了解折叠行为并完善实验。由于应揭示对RNA折叠动态和适应性的新见解，因此结果应广泛影响许多与健康相关的项目。这些发现可能使理性地研究体内稳定性不同的RNA疗法成为可能，并且它们可能会导致对病毒中基因型和表型之间关系的新见解。