The Influence of Secondary Structure on the Folding and Catalysis of Functional RNAs

功能性RNA具有介导活性的复杂三级结构。RNA功能的一个限制是在分子水平上发现的：它的四个碱基的功能多样性很差。特别是，游离核苷酸没有pKa值接近中性，在生物pH值下不能是阳离子。另一方面，组氨酸和赖氨酸具有这些特性，这大大扩展了蛋白质的催化和识别库。如果RNA中缺少这些功能，将极大地限制其功能。然而，一种可能性是，在折叠时，某些A和C的pKa值变得高度扰动并转变为中性和更高。该项目的中心重点是了解二级结构在调节RNA功能特性中的重要性。合作折叠的二级结构基序，这可能具有强烈的位移pKa值，将进行检查。还将探索二级结构折叠影响三级结构功能稳定性的能力;假设在协同展开三级结构的条件下，增加二级结构稳定性将增加功能稳定性。因此，理解协同RNA折叠孤立的二级结构和连接的三级和二级结构贯穿这项研究。最后，在二级结构异常稳定的分子起源将被检查，与稳定性的假设，往往来自静电相互作用。非线性泊松玻尔兹曼（NLPB）计算将被用来确定这样的相互作用，这些将通过模型序列的实验参数化。 更广泛的影响：该项目将整合本科生的研究和出版物的作者。已经制定了一个明确的计划，以确定和招募这些学生在未来的参与研究中代表性不足的群体的学生。该项目还涉及课程改革，将分子概念与数学概念联系起来。一个目标是使学生能够建立简单的分子模型，并从第一原理推导出适当的方程。将为体现这些目标的一系列生物物理问题开发一套模拟程序，并在互联网上提供。

Functional RNAs have intricate tertiary structures that mediate activity. One limitation to RNA function is found at the molecular level: its four bases have poor functional diversity. In particular, free nucleotides do not have pKa's near neutrality and cannot be cationic at biological pH. Histidine and lysine, on the other hand, possess these properties, which greatly expands the catalytic and recognition repertoire of proteins. Absence of such functionalities in RNA would greatly limit its function. One possibility, however, is that upon folding, pKa values for certain A's and C's become highly perturbed and shift to neutrality and beyond. The central focus of this project is on understanding the importance of secondary structure in modulating the functional properties of RNA. Cooperatively folding secondary structural motifs, which may possess strongly shifted pKa values, will be examined. The ability of secondary structure folding to influence the functional stability of tertiary structure will be explored as well; it is hypothesized that under conditions of cooperatively unfolding tertiary structure, increasing secondary structure stability will increase functional stability. Thus, the understanding of cooperativity in RNA folding-of isolated secondary structures and of linked tertiary and secondary structures-pervades this research. Lastly, the molecular origin of exceptional stability in secondary structures will be examined, with the hypothesis that stability often arises from electrostatic interactions. Non-linear Poisson Boltzmann (NLPB) calculations will be used to identify such interactions, and these will be parameterized by experiments on model sequences. Broader Impacts: This project will integrate undergraduates in the research and authorship of publications. A clear plan for identifying and recruiting these students in the future has been developed to involve students from underrepresented groups in the research. The project also involves curriculum reform by connecting molecular concepts and mathematical ones. A goal is to empower students to set up simple molecular models and derive appropriate equations from first principles. A set of simulations for a series of biophysical problems that embody these goals will be developed and made available on the Internet.