Site-specific RNA photocrosslinking of CRISPR-associated and viral RNA-binding proteins using genetic code expansion

Unnatural or non-canonical amino acids (UAAs/ncAAs) allow for the exploration of new functionalities, expanding the molecular biology toolbox. Accordingly, photocrosslinkable UAAs have seen widespread use to study protein-protein interactions, but few studies have explored their RNA crosslinking potential. To address this gap, we expand UAA-RNA crosslinking into a broadly applicable method for structural applications or deducing key interacting sites in genetically encoded site-specific crosslinking immunoprecipitation (GESS-CLIP). p-azido-L-phenylalanine (AzF) was incorporated into CRISPR-associated systems structurally linking the guide RNA, into hepatitis C virus NS3 helicase to photocrosslink viral RNA, and into SARS-CoV-2 unique domain to photocrosslink G-quadruplexes. We then characterized the photocrosslinking ability of the different RBPs while assaying structural integrity and demonstrate effective NS3-genome crosslinking. Furthermore, we demonstrate that crosslinked Cas9-AzF-sgRNA can be transfected into cells to downregulate its genetic target. Taken together, this work offers alternative capabilities for UAA photocrosslinking to support the study and therapeutic development of RBPs. Unnatural amino acids (UAAs) offer novel functionalities, yet their potential for RNA crosslinking remains underexplored. Here, the authors develop a versatile UAA-RNA crosslinking method, demonstrating its application in CRISPR-associated systems and viral proteins.