RNA Folding and Adaptation in a Cellular Context

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结构中。 机械框架。该提案的总体目标是制定全面的 功能性RNA如何在体内折叠的分子机制，并将这些机制与 帮助塑造它们的进化力量。将采取综合办法， 生物物理学和进化驱动力，引起RNA折叠机制， vivo将被识别。第一个具体目标是建立体内RNA的生物物理学原理 通过检查几种天然存在的核糖开关的折叠机制进行折叠， 核酶在模型细胞内和细胞内。第二个具体目标将阐明 进化原理，通过测试RNA的折叠机制来指导RNA在体内的折叠。 将从几个中性漂移选择中出现的序列。因此，无论是自然发生的 和实验室进化的功能性RNA将被检查，总的目标是阐明 RNA在细胞中折叠的一般原理。这项研究涉及开发一系列 模型细胞内RNA的折叠协同性和动力学。在 此外，折叠机制的研究将直接在真核生物和原核生物中进行 细胞使用几种新的方法。进化力量在塑造RNA折叠中的作用 将通过进行几次中性漂移选择来研究体内景观。成员 将评估这些文库的折叠热力学和动力学。的能力 还将评估协同折叠RNA以适应选择压力。方法 在整个研究中应用的方法包括高通量量热法; CD和UV检测 热变性;快速动力学; SAXS;以及表达、结构作图和活细胞 各种细胞中的RNA成像。整个研究过程中的数据将由几个 理论和计算方法，这将有助于理解折叠 行为和完善实验。因为对RNA折叠动力学和 适应应该被揭示，结果应该广泛影响许多不同的健康- 相关项目。这些发现可能使合理地设计RNA疗法成为可能， 不同的体内稳定性，他们可能会导致新的见解之间的关系， 基因型和表型。

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.