Time-Resolved Measurements of Secondary Structure Formation in Single-Stranded Polynucleotides

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The goals of this project are to obtain quantitative information on the intrachain dynamics of single-stranded polynucleotides, and to determine how these dynamics influence the kinetics of secondary structure formation. The kinetics will be monitored using transient absorbance measurements with ~ 10 nanosecond time-resolution after a laser temperature-jump. The experiments are designed to fill a big gap in the folding studies of RNA molecules, namely the time-scales and pathways associated with the collapse of a single-stranded chain and the formation of nucleating hairpins, which occur on time-scales of several nanoseconds-to-microseconds. Previous measurements on hairpin formation have been limited to microsecond time-resolution and have failed to provide a complete picture. In particular, this work will test the hypothesis that transient trapping in misfolded conformations is responsible for reducing the effective diffusion coefficient for intrachain dynamics and for the observed non-Arrhenius temperature dependence of the closing times. Kinetics of hairpin formation will be measured for varying stem sequences designed to significantly enhance the probability of mis-matched stems; the kinetics will also be measured for varying solvent viscosity. A recent statistical mechanical study of the folding/unfolding of a 21-nucleotide RNA hairpin, which included misfolded states in the statistical ensemble, predicted biphasic kinetics with a rapid phase occurring on sub-microseconds. This prediction will be tested. Finally, direct measurements of the first contact time between the two ends of a single-stranded chain will be measured using triplet-triplet energy transfer between a donor and an acceptor attached to the two ends of the chain. To verify the predicted contact times expected for an ideal chain, the measurements will be done under conditions where the self-interactions of the chain are minimized. The contact times relevant for hairpin formation will be measured under solvent conditions that enhance self-interactions and induce local secondary structure.The significance of this study is to develop a quantitative understanding of how a single-stranded polynucleotide organizes itself into simple stable structures and to extend the insights gained from these simple structures to a deeper understanding of the intrachain dynamics and interactions that lead to successively more complex RNA structures. Close comparison between kinetics measurements and statistical mechanical studies of the folding of RNA will provide an ideal ground to test, not only the details of the statistical mechanical models, but also the thermodynamic parameters used for predicting secondary structures in single-stranded DNA and RNA. The primary educational goals are to implement an undergraduate program in Biophysics. A new course for undergraduates "Introduction to Biophysics" will be developed as a way of attracting students into the Biophysics major. A higher level course "Molecule and Cell Biophysics" intended for advanced undergraduates and beginning graduate students was developed two years ago and is now offered each year.