Poster abstracts

Poster number 80 submitted by Eric Patrick

Single-Base Resolution Nucleic Acids Dynamics

Eric M. Patrick (Department of Chemistry, Wayne State University, Detroit MI, 48202), Elvin Aleman (Department of Chemistry, Wayne State University, Detroit MI, 48202), Chamaree de Silva (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus OH, 43210 ), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus OH, 43210 ), David Rueda (Department of Chemistry, Wayne State University, Detroit MI, 48202)

Abstract:
Over the past decade, single-molecule studies with extrinsic fluorescent dyes have provided exciting new insight into nucleic acids folding and accompanying conformational changes. However, the use of ‘semi-intrinsic’ fluorescent nucleotide analogues, such as 2-aminopurine (2AP) and Pyrrolo-C (PC), has not been reported at the single-molecule level despite their high photon rate. For instance, 2-aminopurine has a photon rate of 2 kHz. Numerous ensemble-averaged studies have proven the utility of these probes to decipher mechanistic steps that involve local rearrangements and base flipping.
Here, we present a new single-molecule assay that takes advantage of the fluorescent properties of 2AP and PC to study local base dynamics. We utilize a novel ‘Click’ chemistry based single-molecule immobilization methodology that enables sufficient minimization of background fluorescence to allow single-molecule detection. We have incorporated 2AP into single- and double-stranded DNA molecules and reveal, in real time, local base dynamics. We are also using PC to study similar motion in RNA molecules. Our results show that 2AP and PC fluoresce steadily, and without blinking, when not stacking with neighboring bases. Removing molecular oxygen by degassing the buffers results in long-lived fluorescence emission lasting for minutes. We anticipate that this strategy will provide new insights into the local dynamics of nucleic acids and nucleic acids-protein complexes.

Keywords: 2-aminopurine, Pyrrolo-C, Base dynamics