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.
Large functional RNAs play critical roles in biology. These functions often arise from a complex three-dimensional structure called the tertiary structure. However that tertiary structure is built up from smaller elements called secondary structure, which themselves are composed of sequence (bases) called primary structure. In general, it is easier to predict and control secondary structure than tertiary structure, so we wanted to understand the influence secondary structure has on tertiary structure. This project had as its overarching goal understanding the interplay between RNA secondary structure and tertiary structure. There are several significant outcomes of our studies. First, we found that the molecules that terminate (called closing base pairs) certain secondary structures (called loops) have a common molecular basis for stability. This was discovered through a combination of experiments on RNA molecules as well as calculations. This finding is important because it reveals a molecular commonality in RNA folding. We also found that secondary structure stability controls tertiary structure stability, but only when the folding is cooperative, meaning the secondary and tertiary structures fold together. This was achieved through experiments on model DNAs as well as biological RNAs. Experiments were conducted and kinetic models developed and evaluated. This finding is important because it helps us understand, for example, how life can adapt to changing environmental conditions. Lastly, we discovered rules for how some of the bases in RNA become protonated and positively charged. In so doing, we developed new experimental methodologies for measuring proton binding involving NMR, Raman, and fluorescence detection. These accomplishments are important because such bases serve critical roles in RNA catalysts, gene expression, and virus replication. The broader impacts of this proposal were training of six undergraduates, 5 of whom are women. Three of these students co-authored refereed scientific papers with the PI. All of the undergraduate students are now enrolled in professional schools, either graduate, pharmacy, or medical school. In addition, we hosted middle and high school students for demonstrations on biological research, which involved tours of our laboratory and hands-on isolation of DNA from fruit. The PI also visited the local grade school on a number of occasions, where he engaged 3rd and 4th graders in hands-on experiments and discussions about the natural world.
