Multiplexed single-molecule analysis of barcoded protein variants
Single-molecule methods typically analyse only a few protein variants at a time, limiting high-throughput screening. Here, authors develop a DNA barcoding strategy that enables parallel identification and kinetic profiling of protein variants in a single experiment.
Article Open access Published: 12 October 2026 Nature Communications ( 2026 ) Cite this article We’re sharing this article early to provide faster access to peer-reviewed, accepted research. It is citable and carries a permanent DOI. This version is subject to further edits and will be replaced automatically by the final Version of Record.
All legal disclaimers apply. Abstract Single-molecule techniques provide exceptional resolution of biomolecular dynamics but are limited by low throughput. Here, we present a DNA barcoding strategy that enables parallel kinetic profiling of multiple protein variants in a single experiment.
Each protein is covalently linked to a unique DNA barcode, decoded through transient hybridisation of fluorescent probes distinguished by colour and binding duration. Applied to a library of 16 ClpS variants, this approach revealed ~5-fold variation in dissociation kinetics and uncovered previously unrecognised bimodal binding behaviour arising from distinct structural subpopulations.
By enabling scalable, multiplexed functional characterisation at the single-molecule level, this platform provides a general framework for protein engineering, directed evolution and the development of next-generation single-molecule technologies. Acknowledgements The authors would like to thank members of the Spenkelink, van Oijen, and Griffiths labs for helpful discussions.
Funding This work was supported by the Australian National Health and Medical Research Council (Investigator grant 2007778 to L.M.S.), the Human Frontiers of Science Program (Program grant RGP0021/2020 to A.D.G. and A.M.v.O.), the Australian Research Council (Discovery Project Grant DP240101399 to L.M.S., A.M.v.O. and A.D.G., Centre of Excellence in Quantum Biotechnology CE230100021 to H.Y.).
Author information Authors and Affiliations Molecular Horizons and School of Science, University of Wollongong, Wollongong, NSW, Australia Stefan H. Mueller, David M. Grimson, Holden Paz, Aidan C.
Fitch, Haibo Yu, Antoine M. van Oijen & Lisanne M. Spenkelink ARC Centre of Excellence in Quantum Biotechnology, University of Wollongong, Wollongong, NSW, Australia Haibo Yu Laboratoire de Biochimie, Chimie Biologie et Innovation, ESPCI Paris, Université PSL, Paris, France Marco Ribezzi-Crivellari & Andrew D.
Griffiths Authors Stefan H. Mueller David M. Grimson Holden Paz Aidan C.
Fitch Haibo Yu Marco Ribezzi-Crivellari Andrew D. Griffiths Antoine M. van Oijen Lisanne M. Spenkelink Corresponding author Correspondence to Lisanne M.
Spenkelink . Ethics declarations Competing interests This work was financially supported by Quantum-Si. Antoine van Oijen and Andrew Griffiths were members of the Quantum-Si Scientific Advisory Board.
The remaining authors declare no competing interests. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ . Reprints and permissions About this article Cite this article Mueller, S.H., Grimson, D.M., Paz, H. et al. Multiplexed single-molecule analysis of barcoded protein variants.
Nat Commun (2026). https://doi.org/10.1038/s41467-026-78581-y Download citation Received : 27 February 2026 Accepted : 28 September 2026 Published : 12 October 2026 DOI : https://doi.org/10.1038/s41467-026-78581-y
Article text via FreeNewsAPI. Rights remain with Nature.
Read on publisher site → Opens Nature in a new tab
