The overall goal of the proposed work is to produce a quantitative physical model of how the fluorescent probes and the biopolymer of interest (RNA in this case) each contribute to the observed structural dynamics in both single-molecule and bulk fluorescence-detected resonance energy transfer (FRET) experiments. This will be accomplished through a combination of single-molecule spectroscopy, bulk time-resolved spectroscopy, and molecular dynamics simulations.
The specific aims are: 1. Acquire bulk, time-resolved data. Three RNA motifs will be examined (duplex, Loop A, Loop B) with each of several fluorescent probe pairs (Cy3-Cy5, fluorescein-TMR, Alexa488 - Alexa594). Note that the Loop A and Loop B motifs are portions of the complete hairpin ribozyme to be analyzed in aim 4. 2. Acquire single-molecule FRET data on each system of interest. 3. Model the molecular dynamics of each system computationally. 4. Develop a quantitative physical description of the model RNA systems and apply this to the complete hairpin ribozyme, for which experimental FRET data have already been acquired. 5. Design a single-molecule microscope for use at Hope College, and apply for funding for the instrument. The findings of the proposed research will improve the understanding of FRET measurements, and especially single-molecule FRET measurements, allowing researchers to make more direct connection between the structural dynamics observed experimentally and those of interest in their systems. By applying these results to the hairpin ribozyme, the proposed research will contribute to the development of therapeutic agents for human disease. For instance, because human gene therapy utilizes the hairpin ribozyme for the inactivation of viral and other undesirable RNAs, improved understanding of the structural dynamics of the hairpin ribozyme will facilitate the design of improved ribozymes for therapeutic applications. Relevance: The proposed research will improve the utility of a technique called FRET that is widely used to understand the molecular-level function of proteins, RNA, and DNA. These improvements will be exploited during the grant period to better understand the function of the hairpin ribozyme, which will lead to therapeutic applications such as improved human gene therapy techniques. ? ? ?
| Speelman, Amy L; Munoz-Losa, Aurora; Hinkle, Katie L et al. (2011) Using molecular dynamics and quantum mechanics calculations to model fluorescence observables. J Phys Chem A 115:3997-4008 |
| Muñoz-Losa, Aurora; Curutchet, Carles; Krueger, Brent P et al. (2009) Fretting about FRET: failure of the ideal dipole approximation. Biophys J 96:4779-88 |
