Dosa: A method to covalently barcode proteins for high-throughput biochemistry

Deep mutational scanning couples a protein's activity to DNA sequencing for high-throughput assessment of the effects of all single amino acid substitutions, but it largely uses indirect assays, like cell proliferation, as proxy for protein activity. Here, we covalently link variant proteins in vivo to an RNA barcode by fusing them to Escherichia coli tRNA (m₅U₅₄) methyltransferase TrmA (E358Q). This methyltransferase mutant forms a covalent bond with a tRNA T-arm stem-loop sequence, which we embed in an RNA along with a unique barcode. Following cell lysis, variant proteins are separated in vitro according to their biochemical properties and identified by sequencing their covalently linked barcodes. The in vitro assays can be carried out in highly denaturing conditions, such as 8 M urea, due to the covalent RNA-protein linkage. We use this method, Dosa, to analyze a large pool of FLAG epitope variants for binding to an anti-FLAG-antibody, to profile substrate variants for their cleavage by enteropeptidase and human rhinovirus 3C protease, and to measure the solubility of several hundred Aβ(1-42) variants. This method should be amenable to numerous biochemical assays with proteins produced in E. coli or mammalian cells.