Cryo-EM Structure of R-loop monoclonal antibody S9.6 in recognizing RNA:DNA hybrids

The three-stranded chromatin structure R-loop is commonly found in the genomes of different species, and functions as the double-edged sword in gene expression and genome stability. The monoclonal antibody S9.6 specifically recognizes RNA:DNA hybrids, and has been widely used as a powerful tool to detect R-loops genome-widely. However, the structure basis and the molecular recognition mechanism of S9.6 to the nucleic acid substrates is still limited. Here, applying cryo-electron microscopy, we have determined a 4.9 [A] structure of S9.6 antigen-binding fragment (Fab) complexed with RNA:DNA hybrids. We found that the native Fab cleaved from S9.6 antibody has much higher affinity to RNA:DNA hybrids than to the double-strand RNAs, and the minimum length of the hybrids should be more than 8 base-pair. The structure of Fab binding to the hybrids suggested one loop of S9.6 heavy chain inserts into the minor groove in the RNA:DNA hybrids, and the other three loops flank to the hybrids. The top of four loop all enrich with aromatic or positive charge residues which are potentially responsible for nucleic acids binding specificity. Our results revealed the recognition mechanism of S9.6 on R-loops, which directs the future engineering of S9.6, and thus could further promote R-loop biology studies.