Poster abstracts
Poster number 102 submitted by Golam Mustafa
Short looped DNA as a Force Transducer: Conversion of single molecule FRET signal into force information
Golam Mustafa, William A. Roy (Department of Physics, Kent State University, Kent, OH 44242, United States), Cho-Ying Chuang, Matthew J. Comstock (Department of Physics, Michigan State University, East Lansing, MI 48824, United States), Mohamed M. Farhath, Soumitra Basu (Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242, United States), Nilisha Pokhrel, Edwin Antony (Department of Biological Sciences, Marquette University, Milwaukee, WI 53201, United States), Yue Ma, Kazuo Nagasawa (Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan), Hamza Balci (Department of Physics, Kent State University, Kent, OH 44242, United States)
Abstract:
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.
References:
1. Vafabakhsh, Reza, and Taekjip Ha. "Extreme bendability of DNA less than 100 base pairs long revealed by single-molecule cyclization." Science 337.6098 (2012): 1097-1101.
2. Jeong, Jiyoun, Tung T. Le, and Harold D. Kim. "Single-molecule fluorescence studies on DNA looping." Methods 105 (2016): 34-43.
3. Woodside, Michael T., et al. "Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins." Proceedings of the National Academy of Sciences 103.16 (2006): 6190-6195.
4. Woodside, Michael T., et al. "Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins." Proceedings of the National Academy of Sciences 103.16 (2006): 6190-6195.
Keywords: Single molecule FRET, Force sensor, Looped dsDNA