A Modular DNA Scaffold to Study Protein-Protein Interactions at Single-Molecule Resolution

We introduce junctured-DNA (J-DNA) forceps as a generic platform for real-time observation, at the single-molecule level, of biomolecular interactions. The tool is based on a modular double-strand DNA construct to which proteins of interest can be attached using various tagging strategies. When combined with magnetic tweezers, J-DNA allows us to simultaneously monitor individual interactions on multiple forceps in parallel, and over periods of several days. Interactions with a lifetime ranging from 50 ms to 5000 s can be probed at a constant applied force as low as 30 fN, providing solution-like measurements as well as access to the force-dependence of the interaction. This tool thus provides straightforward and robust access to single-molecule force spectroscopy, in particular for the study of weak interactions. We present experiments characterizing the interactions between rapamycin and the FKBP12 and FRB proteins, a system relevant in both medicine and chemical biology. The zero-force lifetime of the ternary complex is 32.5 ± 0.4 s (SEM) at 21.7 oC and varies with temperature as well as magnitude and direction of the probing force, yielding after analysis the position of the transition state in space and energy as well as the direction of the dissociation axis. We demonstrate the broad applicability of the approach by using it to study the strength and regulation of receptor-scaffold interactions between components of inhibitory synapses, namely the glycine receptor (GlyR) and its scaffolding protein, gephyrin. Our results reveal that the association and dissociation of the cytoplasmic β-loop of the GlyR with a trimer of gephyrin displays a mean interaction lifetime on the order of 10 s, in line with the slow exchange kinetics of the receptor at synapses in living cells.