Multiplexed single-molecule analysis of barcoded protein variants

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.