Multiplexed single-molecule analysis of barcoded protein variants

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.

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BibTeX RIS

BibTeX

@article{mueller2026multiplexed,
  title = {{Multiplexed single-molecule analysis of barcoded protein variants}},
  author = {Stefan H. Mueller and David M. Grimson and Holden Paz and Aidan C. Fitch and Haibo Yu and Marco Ribezzi-Crivellari and Andrew D. Griffiths and Antoine M. van Oijen and Lisanne M. Spenkelink},
  journal = {Nature Communications},
  year = {2026},
  month = oct,
  doi = {10.1038/s41467-026-78581-y},
  url = {https://www.nature.com/articles/s41467-026-78581-y},
  note = {CC BY 4.0},
}

RIS

TY  - JOUR
TI  - Multiplexed single-molecule analysis of barcoded protein variants
AU  - Stefan H. Mueller
AU  - David M. Grimson
AU  - Holden Paz
AU  - Aidan C. Fitch
AU  - Haibo Yu
AU  - Marco Ribezzi-Crivellari
AU  - Andrew D. Griffiths
AU  - Antoine M. van Oijen
AU  - Lisanne M. Spenkelink
PY  - 2026
DA  - 2026/10/12
JO  - Nature Communications
DO  - 10.1038/s41467-026-78581-y
UR  - https://www.nature.com/articles/s41467-026-78581-y
AB  - 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.
SN  - 2041-1723
ER  -

Abstract

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