Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

39-POS Board 39 SHORT LOOPED DNA AS A FORCE TRANSDUCER: CONVERSION OF SINGLE MOLECULE FRET SIGNAL INTO FORCE INFORMATION Golam Mustafa 1 ; Cho-Ying Chuang 2 ; William A Roy 1 ; Mohamed M Farhath 3 ; Nilisha Pokhrel 4 ; Yue Ma 5 ; Kazuo Nagasawa 5 ; Edwin Antony 4 ; Matthew J Comstock 2 ; Soumitra Basu 3 ; Hamza Balci 1 ; 1 Kent State University, Department of Physics, Kent, OH, USA 2 Michigan State University, Department of Physics, East Lansing, MI, USA 3 Kent State University, Department of Chemistry and Biochemistry, Kent, OH, USA 4 Marquette University, Department of Biological Sciences, Milwaukee, WI, USA 5 Tokyo University of Agriculture and Technology, Department of Biotechnology and Life Science, Tokyo, Japan A multiplexed, high throughput single-molecule force sensor and transducer concept has been developed that converts fluorescence signal into force information via single molecule Förster resonance energy transfer (smFRET). A double-stranded DNA (dsDNA) loop has been formed by bridging the ends of a ~100 base pair (bp) long dsDNA with a nucleic acid secondary structure (NAS), such as a hairpin or a G-quadruplex (GQ). The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough. The FRET efficiency between donor and acceptor (D&A) fluorophores placed across the NAS reports on its folding state. As proof-of-principle measurements, 70 bp, 90 bp and 110 bp long dsDNA constructs were bridged by a DNA hairpin and KCl was titrated to change the tension across the DNA hairpin. Later, the interactions of a GQ structure formed by thrombin binding aptamer (TBA) with a destabilizing protein, Replication Protein A (RPA), and a stabilizing small molecule, an oxazole telomestatin derivative, were studied while the TBA-GQ is maintained under tension by a 110-bp long looped dsDNA. The force required to unfold TBA-GQ was independently investigated with high-resolution optical tweezers (OT) measurements that established the relevant force to be a few pN, which is consistent with the force generated by the looped dsDNA. Since hundreds of such molecules could potentially be imaged simultaneously, it is possible to perform high- throughput force measurements with single molecule sensitivity. The proposed method enables studying NAS, protein, and small molecule interactions using a highly-parallel FRET-based assay while the NAS is kept under an approximately constant force.

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