A modular DNA scaffold to study protein–protein interactions at single-molecule resolution

• Published in: Nature nanotechnology Vol. 14; no. 10; pp. 988 - 993
• Main Authors: Kostrz, Dorota, Wayment-Steele, Hannah K., Wang, Jing L., Follenfant, Maryne, Pande, Vijay S., Strick, Terence R., Gosse, Charlie
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.10.2019; Nature Publishing Group
• Subjects: 119/118; 631/61/350; 639/766/747; 639/925/926; Animals; Biophysics; Chemical Sciences; Chemistry and Materials Science; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA, Single-Stranded - chemistry; Energy of dissociation; Humans; Materials Science; Models, Molecular; Nanostructures - chemistry; Nanotechnology; Nanotechnology - methods; Nanotechnology and Microengineering; Organic chemistry; Protein interaction; Protein Interaction Mapping - methods; Proteins; Rapamycin; Scaffolds; Sirolimus - metabolism; Spectroscopy; Tacrolimus Binding Protein 1A - metabolism; TOR Serine-Threonine Kinases - metabolism
• ISSN: 1748-3387; 1748-3395
• DOI: 10.1038/s41565-019-0542-7

The residence time of a drug on its target has been suggested as a more pertinent metric of therapeutic efficacy than the traditionally used affinity constant. Here, we introduce junctured-DNA tweezers as a generic platform that enables real-time observation, at the single-molecule level, of biomolecular interactions. This tool corresponds to a double-strand DNA scaffold that can be nanomanipulated and on which proteins of interest can be engrafted thanks to widely used genetic tagging strategies. Thus, junctured-DNA tweezers allow a straightforward and robust access to single-molecule force spectroscopy in drug discovery, and more generally in biophysics. Proof-of-principle experiments are provided for the rapamycin-mediated association between FKBP12 and FRB, a system relevant in both medicine and chemical biology. Individual interactions were monitored under a range of applied forces and temperatures, yielding after analysis the characteristic features of the energy profile along the dissociation landscape. A double-strand DNA scaffold enables real-time observations of protein–protein interactions at the single-molecule level.