RNA Folding and Adaptation in a Cellular Context

描述（由申请人提供）：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制框架中。本提案的主要目标是发展功能性rna如何在体内折叠的综合分子机制，并将这些机制与帮助塑造它们的进化力量联系起来。将采取一种综合的方法，其中生物物理和进化驱动力产生RNA折叠机制在体内将被确定。第一个具体目标是通过研究模型细胞质和细胞中几种自然发生的核开关和核酶的折叠机制，建立体内RNA折叠的生物物理原理。第二个具体目标将阐明进化原则，指导RNA折叠在体内通过测试折叠机制的序列，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都将被检查，其总体目标是阐明细胞中RNA折叠的一般原理。该研究包括开发一系列模型细胞质，并测试rna在这些模型细胞质中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进行。进化力量在体内形成RNA折叠景观中的作用将通过进行几个中性漂移选择来研究。这些库的成员将被评估折叠热力学和动力学。合作折叠rna适应选择压力的能力也将被评估。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;粉煤灰;以及各种细胞中rna的表达、结构定位和活细胞成像。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并改进实验。由于RNA折叠动力学和适应性的新见解将被揭示，结果将广泛影响许多不同的健康相关项目。这些发现可能使合理设计具有不同体内稳定性的RNA疗法成为可能，并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.