Single molecule fluorescence studies of biomolecular interactions
Single molecule fluorescent techniques have become standard approaches to study protein-DNA interactions. However, these techniques have largely been confined by limitations in assays to studying the interaction between simple DNA substrates and a single protein. During my PhD I developed several novel assays to study a long-standing controversial biophysics question (flexibility of short dsDNA), genome packaging in viruses (Influenza and T4) and dynamics of challenging protein complexes (membrane proteins). The classical view of DNA posits that DNA must be stiff below the persistence length (<150 base pair) but recent studies addressing this have yielded contradictory results. We developed a fluorescence-based, protein-free, assay for studying the cyclization of single DNA molecules in real time. The looping rate for short DNA molecules has remarkably weak length dependence between 67 and 106 bps, deviating significantly from the worm-like chain model. We propose that many biologically significant protein-DNA interactions that involve looping and bending of DNA below 100 bp likely use this intrinsic bendability of DNA. One of the critical aspects of a virus life cycle is packaging of the viral genome. Different viruses have devised intelligent mechanisms to perform this task. I studied packaging mechanism in Bacteriophage T4 and Influenza. Influenza A virus possesses a segmented genome of eight, single-stranded RNAs. However, the exact copy number of each viral RNA segment per individual virus particles has been controversial for the past 50 years. To address this question we combined single molecule TIRF microscopy and multi-color fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Our results showed that a high percentage of virus particles package a single copy of each segment of viral RNAs. Our findings support a model that the packaging of influenza genome is a selective and robust process. Finally we developed a single molecule fluorescence assay to study initiation and re-initiation of dsDNA packaging in the T4 bacteriophage. Using this assay we quantified the details of T4 “packasome” assembly. Also, we showed that the T4 packaging machine can package multiple DNA into the same head in burst-like fashion.
Single molecule, Fluorescence Resonance Energy Transfer (FRET), Deoxyribonucleic Acid (DNA), Influenza, bacteriophage T4 packaging
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign