RNA Folding and Adaptation in a Cellular Context

细胞环境中的 RNA 折叠和适应

• 批准号: 8671799
• 负责人: PHILIP C BEVILACQUA
• 金额: $ 26.42万
• 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/8671799
• 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; Functional RNA; 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; Variant; Virus; aptamer; base; biophysical properties; cellular imaging; design; driving force; experience; in vivo; insight; interest; member; next generation sequencing; novel; novel strategies; pressure; public health relevance; research study; temperature jump; therapeutic development

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.

RNA在细胞中的折叠还没有被很好地理解，也没有被整合到一个有凝聚力的 机械论框架。这项提案的广泛目标是制定全面的 功能RNA在体内折叠的分子机制及其与 帮助塑造它们的进化力量。将采取综合方法，在 引起核糖核酸折叠机制的生物物理和进化驱动力 VIVO将会被确认。第一个具体目标是建立体内rna的生物物理原理。 通过研究几种自然产生的核糖开关的折叠机制和 核酶存在于模型细胞质和细胞内。第二个具体目标将阐明 通过测试RNA的折叠机制来指导体内RNA折叠的进化原理 将从几个中性漂移选择中产生的序列。因此，两者都是自然发生的 实验室进化的功能性RNA将被研究，总体目标是阐明 RNA在细胞内折叠的一般原则。这项研究涉及开发一系列 建立细胞质模型，并测试其中RNA的折叠协同性和动力学。在……里面 此外，折叠机制的研究将直接在真核和原核生物中进行。 细胞使用几种新的方法。进化力量在塑造RNA折叠中的作用 活体中的风景将通过进行几次中性漂移选择来研究。成员： 将对这些文库进行折叠热力学和动力学评估。的能力 还将评估以合作方式折叠RNA以适应选择压力。方法，以 在整个研究中的应用包括高通量量热法；CD和UV检测 热变性；快速动力学；SAXS；表达、结构作图和活细胞 对各种细胞中的RNA进行成像。整个研究过程中的数据将由几个 理论和计算方法，这将被用来帮助理解折叠 行为和改进实验。因为对RNA折叠动力学的新见解和 适应应该被揭示，结果应该广泛地影响许多不同的健康- 相关项目。这一发现可能使合理设计RNA疗法成为可能 不同的体内稳定性，它们可能导致对之间关系的新见解 病毒的基因型别和表型。