Single-molecule methods access biomolecular distributions, transient states, and asynchronous dynamics inaccessible to standard ensemble techniques. Although extremely powerful, fluorescence-based single-molecule detection platforms are currently limited by the lack of high-throughput methods for the generation and screening of large libraries of dye-labeled proteins. Here, we demonstrate proof-of-principle that by combining purified and reconstituted in-vitro translation, quantitative unnatural amino acid incorporation via sense codon reassignment, and either strain-promoted or copper-catalyzed azide-alkyne cycloaddition we can overcome these bottlenecks. We present a purification-free and parallelizable in-vitro approach to generating dual-labeled proteins and ribosome-nascent-chain (RNC) libraries suitable for single-molecule FRET-based structural phenotyping. Importantly, dual-labeled RNC libraries enable single molecule co-localization of genotypes with phenotypes, and thus multiplexed single molecule screening of protein libraries. Such an approach to high-throughput single molecule screening may be useful for the in-vitro directed evolution of proteins with designer single molecule phenotypes.

Pushing click chemistry to its limits: An in-vitro sample generation pipeline for high-throughput single-molecule FRET-based screening of proteins and ribosome-bound nascent chain libraries

253rd National Meeting (2017)

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