Specific Recognition of a Single-Stranded RNA Sequence by a Synthetic Antibody Fragment. Issue 20 (9th October 2016)

• Record Type: Journal Article
• Authors: Shao, Yaming; Huang, Hao; Qin, Daoming; Li, Nan-Sheng; Koide, Akiko; Staley, Jonathan P.; Koide, Shohei; Kossiakoff, Anthony A.; Piccirilli, Joseph A.
• Is Part Of: Journal of molecular biology. Volume 428:Issue 20(2016:Oct. 15)
• Page Start: 4100
• Page End: 4114
• Publication Date: 2016-10-09
• Subjects: CDR complementarity-determining region -- ncRNA non-coding RNA -- ssRNA single-stranded RNA -- Deoxy R deoxynucleotide version of the R strand -- MR molecular replacement -- PBS phosphate-buffered saline -- R.T. room temperature; Phage display -- RNA-binding Fab -- Crystal structure -- ssRNA -- YSGR library; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2016.08.029
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: 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 the engineering of effective Fab libraries guided by the knowledge of the principles that govern RNA recognition. Here, we describe a Fab identified from a 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 (ssRNA) 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 2.38 Å. The Fab traps the RNA in a hairpin conformation that contains a 2-bp duplex capped by a tetraloop. The paratope surface consists of residues located in four complementarity-determining regions 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 the recognition of ssRNA via a stem–loop conformation, together with our two previous structures involving the recognition of an RNA hairpin loop and an RNA tertiary structure, reveals the capacity of minimalist libraries biased with tyrosine, serine, glycine, and arginine to form binding surfaces for specific RNA conformations and distinct levels of RNA structural hierarchy. Graphical Abstract: Highlights: Fabs that bind RNA have great potential as biomedical research tools. Fab BRG binds to a 12-nt ssRNA with high affinity and specificity. The Fab BRG stabilizes a hairpin conformation of the RNA. Complementarity-determining region-H3 and tyrosine play dominant roles in RNA recognition. Fab provides a versatile scaffold for RNA binding.