2008 Rustbelt RNA Meeting
In vivo, many RNA molecules can adopt multiple conformations depending on their biological context. For example, an RNA molecule that is initially in a stable hairpin conformation will at a later stage of its biological cycle interact with a second RNA molecule, which in turn will trigger a dimerization reaction of the two molecules. This is the case of the HIV Dimerization Initiation Sequence (DIS) or the DsrA RNA in bacteria. It is quite common that the initial interaction between the two RNAs takes place via complementary unpaired regions, thus forming a so-called kissing complex. However, the exact kinetic mechanism by which the two RNA molecules reach the dimerized state is still not well understood.
To investigate the refolding energy surface of RNA molecules, we have developed new technology based on the combination of single molecule spectroscopy with laser induced temperature jump kinetics, called Laser Assisted Single-molecule Refolding (LASR). LASR enables us to induce folding reactions of otherwise kinetically trapped RNAs at the single molecule level, and to characterize their folding landscape. Single molecule time trajectories show that we can drive the dimerization reaction between two trapped kissing RNA hairpins with LASR, and use this data to calculate folding enthalpies and entropies. Our LASR experiments indicate that RNA kissing complex is a stable intermediate state that facilitates the dimerization reaction.
LASR provides an exciting new approach to study molecular memory effects and kinetically trapped RNAs in general. LASR should be readily applicable to study DNA and protein folding as well.
Keywords: Single molecule, refolding, RNA