Document Type

Article

Language

eng

Publication Date

12-15-2018

Publisher

Elsevier

Source Publication

Biosensors and Bioelectronics

Source ISSN

0956-5663

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.

Comments

Accepted version. Biosensors and Bioelectronics, Vol. 121 (December 15, 2018): 34-40. DOI. © 2018 Elsevier B.V. Used with permission.

Available for download on Tuesday, December 15, 2020

antony_12622acc.docx (472 kB)
ADA accessible version

Included in

Biology Commons

Share

COinS