Abstract
Double-stranded (ds) RNAs are major structural components of the transcriptome, hallmarks of viral infection, and primary triggers of innate immune responses. The J2 monoclonal antibody is the gold-standard method to discover and map endogenous dsRNAs across subcellular locations and cell surfaces, detect exogenous RNAs in viral infection, and surveil mRNA prophylactics and therapeutics for inflammatory dsRNAs. To define its epitope, specificity, and mechanism, we determine a 2.85 Å co-crystal structure of J2 antigen-binding fragment (Fab) bound to dsRNA. J2 uses its heavy and light chains in tandem to track the dsRNA minor groove, recognizing a staggered 8-bp duplex. J2 is exquisitely selective for dsRNAs, requires 14 bp for robust binding, and exhibits greatly diminished binding for GC-rich dsRNAs. J2 and R-loop-specific S9.6 antibody share a common recognition strategy distinct from intracellular dsRNA-binding proteins. This study provides mechanistic insights into dsRNA recognition and establishes a framework for reliable application and data interpretation of the J2 antibody in RNA discovery.