A force sensor that converts fluorescence signal into force measurement utilizing short looped DNA

Abstract

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.

Determinants of cyclization–decyclization kinetics of short DNA with sticky ends

“…This stress can affect the binding affinity of the protein complexes, and thereby alter the lifetime of the looped state [6,11,12]. Recently, small DNA loops have also been used as force sensors and applicators to study bending mechanics of DNA itself or force-dependent conformational changes of other biomolecules [13][14][15][16][17]. Therefore, measuring looping and unlooping dynamics of short DNA segments can give us insights into the energetics and internal forces that govern loop-associated processes and applications.…”

Weak tension accelerates hybridization and dehybridization of short oligonucleotides

“…The measured binding ( k on ) and unbinding ( k off ) rate constants thus reflect hybridization and dehybridization transitions of a short DNA homoduplex or DNA/RNA heteroduplex. Our DNA bow assay exploits the bending rigidity of dsDNA to generate small forces and is conceptually similar to the force clamp implemented with DNA origami [59] and a loopbased force transducer [60]. An identical DNA construct has also been used in other studies [61, 62].…”

Weak tension accelerates hybridization and dehybridization of short oligonucleotides

“…The measured binding (k on ) and unbinding (k off ) rate constants thus reflect hybridization and dehybridization transitions of a short DNA homoduplex or DNA/RNA heteroduplex. Our DNA bow assay exploits the bending rigidity of dsDNA to generate small forces and is conceptually similar to the force clamp implemented with DNA origami [59] and a loopbased force transducer [60]. An identical DNA construct has also been used in other studies [61,62] S7).…”