Open Access Dissertation
Date of Award
Non-coding RNAs must fold into precise secondary and tertiary structures in order to perform the biological functions. Due to the flexibility of RNA, the RNA folding energy landscape can be rugged and full of local minimum (kinetic trap). To provide a means to study kinetically trapped RNAs, we have developed a new technique combining single-molecule FRET detection with laser induced temperature jump. We have calibrated the magnitude of the temperature jump with 1˚C accuracy using gold micro-size sensor. The accuracy of temperature calibration was confirmed by close agreement between single-molecule and bulk DNA duplex melting experiments.
HIV 1 DIS RNAs form a kissing complex in the loop region and proceed to the extended duplex structure with the help of enzymes or other cofactors in the later stage of viral replication. The kissing complex itself is very stable, which makes it a unique optimal model system to study kinetically trapped RNAs. The application of LASR to kissing hairpins has allowed us, for the first time, to drive a molecular reaction and monitor the process at the single molecule level. The melting curve for the dissociation and dimerization was used to estimate the thermodynamic properties of the reaction, such as melting temperature, cooperativity, and the enthalpy change. Mutational studies have allowed dissection of the contribution of base pairs to kissing complex stability. And the ratio of the competing reaction pathways was determined.
LASR experiments designed to the study of the origin of the memory effect in the hairpin ribozyme folding introduced inter-conversions between the subpopulations of hairpin ribozyme with distinct undocking kinetic rates. This provides strong evidence for the hypothesis that the memory effect is an intrinsic property of hairpin ribozyme folding. Eyring analysis was adopted to fit the enthalpic and entropic height of the inter-conversion barrier. Our results suggest that for inter-conversion to occur, surprisingly, a small number of interactions are broken. However inter-conversion is rare due to a large negative entropic term. Negative entropy indicates a rigid transition state for inter-conversion and an increased free energy barrier height with increased temperature. This explains why there are few inter-conversions even at high temperatures such as 78 ˚C. The entropic barrier may primarily arise from the stretching of the S-turn.
Zhao, Rui, "Laser-assisted single-molecule refolding" (2011). Wayne State University Dissertations. Paper 266.