An In Vitro Sample Generation Pipeline For High-Throughput Single-Molecule Fret Based Screening Of Proteins

Single-molecule methods access biomolecular distributions, transient states, and asynchronous dynamics inaccessible to standard ensemble techniques. Although extremely powerful, the ability to screen large biomolecular libraries using fluorescence-based single-molecule detection platforms is still a challenge due to 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 (e.g. using zero-mode-waveguides). 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.