Specific Recognition of a Single-stranded RNA Sequence by a Synthetic Antibody Fragment

摘要

Antibodies that bind RNA represent an unrealized source of reagents for synthetic biology and for characterizing cellular transcriptomes. However, facile access to RNA binding antibodies requires engineering of effective Fab libraries guided by knowledge of the principles that govern RNA recognition. Here we describe a Fab identified from a YSGR minimalist synthetic library during phage display against a branched RNA target. The Fab (BRG) binds with 20 nM dissociation constant to a single-stranded RNA sequence adjacent to the branch site and can block the action of debranchase enzyme. We report the crystal structure in complex with RNA target at 2.38Å. The Fab traps the RNA in a hairpin conformation that contains a two base-pair duplex capped by a tetraloop. The paratope surface consists of residues located in four CDRs including a major contribution from H3, which adopts a helical structure that projects into a deep, wide groove formed by the RNA. The amino acid composition of the paratope reflects the library diversity, consisting mostly of tyrosine and serine residues and a small, but significant contribution from a single arginine residue. This structure, involving recognition of single-stranded RNA via a stem-loop conformation, together with our two previous structures involving recognition of an RNA hairpin loop and an RNA tertiary structure, reveals the capacity of YSG(R)-biased, minimalist libraries to form binding surfaces for specific RNA conformations and distinct levels of RNA structural hierarchy.