RNA Folding and Adaptation in a Cellular Context

描述（由申请人提供）：RNA在细胞中的折叠尚未得到很好的理解，也没有被整合到一个有凝聚力的机制框架中。该提案的广泛目标是开发功能性RNA如何在体内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。将采取一个全面的方法，其中生物物理和进化的驱动力，在体内产生RNA折叠机制将被确定。第一个具体的目标将建立生物物理学原则，在体内RNA折叠，通过检查几个自然发生的核糖开关和核酶在两个模型细胞和细胞中的折叠机制。第二个具体目标将阐明进化的原则，指导RNA折叠在体内通过测试的折叠机制的序列，将出现从几个中性漂移选择。因此，自然发生的和实验室进化的功能性RNA将被检查，阐明RNA在细胞中折叠的一般原则的总体目标。该研究包括开发一系列模型细胞，并测试这些细胞中RNA的折叠协同性和动力学。此外，折叠机制的研究将直接在真核和原核细胞中使用几个新的方法进行。通过进行几次中性漂移选择，将研究进化力量在体内塑造RNA折叠景观中的作用。这些库的成员将被评估的折叠热力学和动力学。还将评估协同折叠RNA适应选择压力的能力。在整个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; SAXS;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，这些方法将用于帮助理解折叠行为和改进实验。由于对RNA折叠动力学和适应性的新见解应该被揭示，因此结果应该广泛影响许多不同的健康相关项目。这些发现可能使合理地设计具有不同体内稳定性的RNA疗法成为可能，并且它们可能导致对病毒基因型和表型之间关系的新见解。

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.