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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.
Single-molecule fluorescence microscopy is a growing and powerful tool for studying molecular biophysics. However, the field faces several barriers to entry, limiting its uptake. Open-source microscopy is an emerging field where published setups seek to address the barriers to microscopy entry, including single-molecule fluorescence microscopy.
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