Exploring the conformational dynamics of functional RNA molecules by ultrafast time-resolved spectroscopy

RNA has the intrinsic property to adopt multiple conformations through very fast independent as well as coupled motions. Resolving the high-resolution structures of dynamically inter-converting states is a major challenge for traditional biophysical techniques such as NMR spectroscopy and X-ray crystallography which provide an averaged structure of well folded conformations. It has been shown that the presence of multiple conformations contributes to the ruggedness of the RNA energy landscape, and the functionally relevant conformations could be less populated or transiently present in the ensemble of structure. In two different RNA molecules; a ribozyme with an asymmetric internal loop and a protein binding RNA with a trinucleotide bulge, the conformational dynamics and the structure-function correlation are explored by time-resolved ultrafast fluorescence spectroscopy. Here, RNAs are labeled with one of the most extensively studied fluorescent nucleic acid probes 2-aminopurine, to probe the base stacking patterns in specific motifs in RNA. Base stacking interactions are the major stabilizing force in RNA and the base stacking motions contribute to the local dynamics in RNA. Chapter 1 reviews the broad picture of RNA beyond the static structures and the role of conformational dynamics in defining structure-function relationships in RNA. Chapter 2 explains the basic principles and experimental set-up of ultrafast spectroscopy approach used in our lab and a brief discussion of other spectroscopic techniques that complement the time-resolved spectroscopy. Chapter 3 discusses the conformational dynamics and heterogeneity of leadzyme. We observed a highly unique asymmetric loop structure in leadzyme that lacks orderliness in the catalytic loop. Chapter 4 reviews the heterogeneity of HIV-1 TAR RNA, where we probe the free and ligand bound TAR to capture any bound conformation in the free state that can lead to the proposal of a possible conformational capture mechanism in TAR-Tat recognition. Chapter 5 summarizes our findings and the future prospects of employing time-resolved approach to study the dynamics of biomolecules. In conclusion, we observed RNA as an ensemble of structures in different structural context rather than a single rigid static structure.