Engineering site-specific nucleic acid-protein conjugates by utilizing a natural RNAylation reaction

Nucleic acid-protein conjugates are valuable for synthetic biology, therapeutics, and nanotechnology, but current methods often lack site specificity and rely on non-natural linkages. RNAylation, a one-step enzymatic reaction catalyzed by the bacteriophage T4 enzyme ModB where first discovered in vivo during phage infection, enables site-specific conjugation of nucleic acids to proteins via a natural N-glycosidic bond. Here, we establish RNAylation as a novel and robust in vitro platform for generating nucleic acid-protein conjugates, overcoming key limitations of existing strategies. We define design principles for this approach, demonstrate enhanced nucleic acid stability in human cell lysates, and develop an efficient purification workflow. Furthermore, we achieve successful delivery of purified conjugates into human cells, highlighting the potential for functional in vivo applications. Our work expands RNAylation from a phage-specific phenomenon to a versatile, biologically relevant strategy with broad biotechnological potential.