Repurposing Engineered Aminoacyl-tRNA Synthetases for Proximity-Dependent Protein Labeling through the Release of Aminoacyl-AMP Intermediate.

Over the past two decades, engineering efforts have yielded aminoacyl-tRNA synthetases (aaRSs) capable of charging diverse noncanonical amino acids (ncAAs). Here, we report an unexpected and exploitable activity of these engineered aaRSs. In the absence of their cognate tRNA, aaRSs can release the ncAA-AMP intermediate, which labels proximal proteins via covalent acylation. Multiple different aaRSs exhibit this behavior, enabling both self-labeling and trans-labeling of proteins with ncAAs in vitro and in living cells. When using bioorthogonal ncAAs, the resulting labeled proteins can be further functionalized through bioorthogonal conjugation with a fluorophore for visualization or biotin for enrichment. Although labeling by existing aaRSs is slow, we provide preliminary evidence that improved activity is possible through further engineering. Taken together, these results establish a new potential approach for proximity-based protein labeling, analogous to BioID and TurboID. Many aaRSs are available to charge diverse ncAAs, creating opportunities for multiplexed labeling; i.e., different aaRS/ncAA pairs used simultaneously to probe multiple spatially/temporally distinct microenvironments.