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Single-molecule mechanical fingerprinting with DNA nanoswitch calipers

Abstract

Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have substantially affected clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Towards this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA nanoswitch caliper capable of measuring multiple coordinates on single biomolecules with atomic resolution. Using optical tweezers, we demonstrate absolute distance measurements with ångström-level precision for both DNA and peptides, and using multiplexed magnetic tweezers, we demonstrate quantification of relative abundance in mixed samples. Measuring distances between DNA-labelled residues, we perform single-molecule fingerprinting of synthetic and natural peptides, and show discrimination, within a heterogeneous population, between different posttranslational modifications. DNA nanoswitch calipers are a powerful and accessible tool for characterizing distances within nanoscale complexes that will enable new applications in fields such as single-molecule proteomics.

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Fig. 1: Schematic overview of single-molecule mechanical fingerprinting with DNCs.
Fig. 2: Characterization of DNCs with ssDNA targets.
Fig. 3: Calibration of DNCs for peptide targets.
Fig. 4: Single-molecule peptide fingerprinting.
Fig. 5: Single-molecule mechanical fingerprinting of posttranslational modifications in a heterogeneous mixture of peptides.
Fig. 6: Multiplexed single-molecule mechanical fingerprinting of synthetic peptides in different heterogeneous mixtures.

Data availability

Data supporting the findings of this paper are available from the supplementary files and the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

Code used to analyse data in this paper is available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank S. Buratowski, M. Bao and all members of the Wong and Shih Laboratories for helpful discussions and comments on the paper. This work was funded by support from ONR award no. N000141510073, Smith Family Foundation Odyssey Award, grant no. NIH NIGMS R35 GM119537 (W.P.W.) and the Wyss Institute at Harvard. E.K. acknowledges support from the Human Frontier Science Program (grant no. LT001077/2015-C).

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W.P.W. and W.M.S. conceived the project. P.S., T.E.T., D.Y., W.P.W. and W.M.S. designed the experiments. J.I.M. conducted experiments to label peptides with the DNA handles. P.S. conducted experiments with dual-trap optical tweezers. D.Y., P.S. and T.E.T. conducted experiments with magnetic tweezers. D.Y., P.S., H.T.B. and W.P.W. performed data analysis. A.W., E.K., S.C., Y.L., B.N. and A.J.-B. contributed to early experiments. All authors discussed the results and analysis and contributed to the paper, with the initial draft written by P.S. and W.P.W.

Corresponding authors

Correspondence to William M. Shih or Wesley P. Wong.

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W.M.S. and W.P.W. have filed patent applications for various aspects of this work.

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

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Shrestha, P., Yang, D., Tomov, T.E. et al. Single-molecule mechanical fingerprinting with DNA nanoswitch calipers. Nat. Nanotechnol. 16, 1362–1370 (2021). https://doi.org/10.1038/s41565-021-00979-0

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