Fluorescence competition assay measurements of thermodynamics for RNA pseudoknots and multibranch loops

Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2011.RNA thermodynamic stability is a key to predicting RNA structure from sequence and to understanding their functions. RNA pseudoknots and multibranch loops are two important motifs whose thermodynamic properties have not been extensively studied. The recent research progress on RNA pseudoknot folding and finding has been reviewed in this work. Traditional methods, such as UV-melting and Differential Scanning Calorimetry, for measuring RNA thermodynamics are restricted to temperature ranges around the melting temperature for a pseudoknot. In this work, we measured RNA pseudoknot free energy changes at 37 ºC measured by Fluorescence Competition Assays. Sequence dependent studies for the Loop 1-Stem 2 region reveal: (1) the Individual Nearest Neighbor-Hydrogen Bonding (INN-HB) model provides a reasonable estimate for the free energy change when a Watson-Crick base pair in Stem 2 is changed; (2) the loop entropy can be estimated by a Statistical Polymer Model, although some penalty for certain loop sequences is necessary; (3) tertiary interactions can significantly stabilize pseudoknots, and extending the length of Stem 2 may alter tertiary interactions such that the INN-HB model does not predict the net effect of adding a base pair. The results can inform writing of algorithms for predicting and/or designing RNA secondary structures. Computer algorithms such as RNAstructure and MFOLD do not consider the identities of more than 2 unpaired nucleotides in multibranch loops when predicting secondary structure. There is limited experimental data, however, to parameterize this aspect of these algorithms. In this study, UV optical melting and a fluorescence competition assay are used to measure stabilities of multibranch loops containing up to five unpaired adenosines or uridines or a loop E motif. These results provide a test of our understanding of the factors affecting multibranch loop stability and provide revised parameters for predicting stability. The results should help improve predictions of RNA secondary structure