Biosensors and Bioelectronics
A force sensor concept is presented where fluorescence signal is converted into force information via single-molecule Förster resonance energy transfer (smFRET). The basic design of the sensor is a ~100 base pair (bp) long double stranded DNA (dsDNA) that is restricted to a looped conformation by a nucleic acid secondary structure (NAS) that bridges its ends. 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. Three dsDNA constructs with different lengths were bridged by a DNA hairpin and KCl was titrated to change the applied force. After these proof-of-principle measurements, one of the dsDNA constructs was used to maintain the G-quadruplex (GQ) construct formed by thrombin binding aptamer (TBA) under tension while it interacted with a destabilizing protein and stabilizing small molecule. 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. The proposed method is particularly promising as it 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.
Mustafa, Golam; Chuang, Cho-Ying; Roy, William A.; Farhath, Mohamed M.; Pokhrel, Nilisha; Ma, Yue; Nagawawa, Kazuo; Antony, Edwin; Comstock, Matthew J.; Basu, Soumitra; and Balci, Hamza, "A Force Sensor that Converts Fluorescence Signal into Force Measurement Utilizing Short Looped DNA" (2018). Biological Sciences Faculty Research and Publications. 668.
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