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

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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.