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

Abstract

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.

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Fig. 1: Structure of the J-DNA tweezers and first generic strategy used to attach a given protein at a given tip.
Fig. 2: Experimental design and initial results on a single molecule.
Fig. 3: Influence of force and temperature on the dissociation of the FKBP12–rapamycin–FRB complex.
Fig. 4: Influence of the pulling direction on dissociation of the FKBP12–rapamycin–FRB complex.
Fig. 5: Second generic strategy used to engraft a given protein at a given tip of the J-DNA tweezers.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

This work was supported by the grants T-DropTwo (Labex NanoSaclay), T-DropThree (Idex Paris-Saclay), NanoRep (PSL University), Gephyrip (Labex Memolife) and J-DNA (PSL-Valorisation). T.R.S. is part of the ‘Equipe Labellisée’ program of the Ligue Nationale Contre la Cancer. H.K.W.-S. and J.L.W., respectively, acknowledge the National Science Foundation and the China Scholarship Council for PhD fellowships. We thank A. Thomas and L. Friedman for the preliminary experiments, as well as the groups of J. Yan and F. Hausch for discussions on unpublished data.

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D.K., T.R.S. and C.G. conceived the experiments, J.L.W. contributed unique reagents, D.K. and M.F. prepared reagents and carried out measurements, D.K. and T.R.S. carried out primary data analysis, D.K., H.K.W.-S., V.S.P., T.R.S. and C.G. carried out advanced data analysis and modelling and D.K., T.R.S. and C.G. wrote the paper.

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Correspondence to Terence R. Strick or Charlie Gosse.

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Peer review information Nature Nanotechnology thanks Michael Schlierf and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Figs. 1–9, Tables 1 and 2, Methods and Refs. 1–25.

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Kostrz, D., Wayment-Steele, H.K., Wang, J.L. et al. A modular DNA scaffold to study protein–protein interactions at single-molecule resolution. Nat. Nanotechnol. 14, 988–993 (2019). https://doi.org/10.1038/s41565-019-0542-7

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