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. 2016 Oct 9;428(20):4100-4114.
doi: 10.1016/j.jmb.2016.08.029. Epub 2016 Sep 2.

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

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Free PMC article

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

Yaming Shao et al. J Mol Biol. .
Free PMC article

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 20nM 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 2.38Å. 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.

Keywords: Crystal structure; Phage display; RNA-binding Fab; YSGR library; ssRNA.

Figures

Figure 1
Figure 1
The YBL059W branched RNA target and binding by Fabs selected from synthetic phage-displayed Fab libraries. (a). Sequence and structure of the target. The RNA is composed of two strands, L (red) and R (blue), connected by a chemical bond between 5’-end of R and A18 2’-OH of L. The RNA contains a biotin moiety on the 3’-end of L for antigen immobilization during selection. (b). CDR sequences of three selected Fab clones (Fab-BRG, -BRK1, and –BRK2. Only positions with designed diversity are colored according to amino acid type. (c). Fab binding to branched RNA revealed by filter binding assays. Fraction bound reflects the fraction of RNA retained on a nitrocellulose filter as a result of incubation with the indicated Fab. For Fab-BRG, a fit of the data to a binding equation gave KD = 21 ± 3 nM. All binding assays contained PBS at pH 7.4, 0.2 mM EDTA (d). BRG and BRK1 binding to Deoxy R.
Figure 2
Figure 2
Location of the Fab-BRG binding site within the branched RNA. (a). Fab BRG binds strand R with full affinity (KD = 26 ± 1 nM). (b). Fab BRG inhibits Dbr1 debranching of the RNA target ([Dbr1] = 1.4 nM). See Supplementary Fig. 1 for original gel figures. (c). Hydroxyl radical footprinting on strand R in the presence and absence of Fab BRG. Input – control samples without Fe-EDTA treatment; T1 Digestion – ladder generated by treating samples with ribonuclease T1. (d). Fab BRG binds a 10-mer oligonucleotide (A3 to C12) with full affinity (KD = 10 ± 2 nM). (e). Fab BRG binds 3 of the 10 scanning mutagenesis 10-mer constructs with nearly full affinity (See Table 1 for more details).
Figure 3
Figure 3
Crystal structure of the Fab-BRG:R-12 RNA complex. (a). Packing of the Fab-RNA complex (Fab: green; RNA: orange) in the crystal lattice with a unit cell displayed. Symmetry-related molecules are shown in grey. (b). Overall structure of the Fab-RNA complex (Color code: blue – RNA, cyan –CDR-L2, orange – CDR-L3, red – CDR-H1, green – CDR-H2, purple – CDR-H3). RNA is contoured with the |Fobs|-|Fcal| difference map (green mesh, 3.5σ) from a simulated annealing refinement omitting the R-12 RNA. (c). Schematic summary of Fab-RNA interactions on R-12 hairpin. Rectangles represent nucleobases and ovals represent CDR residues colored as in (d). (d). Molecular surface of the Fab-BRG variable domain in complex with the RNA. (e). Interaction between the 12mer RNA and the CDR residues. Orange spheres represent water molecules that mediate hydrogen bonds. Green dashes represent hydrogen bonds, and the red dash represents a salt bridge. Colors correspond to amino acid types (Tyr: yellow, Gly: green, Ser: red, Arg: blue, and others: magenta).
Figure 3
Figure 3
Crystal structure of the Fab-BRG:R-12 RNA complex. (a). Packing of the Fab-RNA complex (Fab: green; RNA: orange) in the crystal lattice with a unit cell displayed. Symmetry-related molecules are shown in grey. (b). Overall structure of the Fab-RNA complex (Color code: blue – RNA, cyan –CDR-L2, orange – CDR-L3, red – CDR-H1, green – CDR-H2, purple – CDR-H3). RNA is contoured with the |Fobs|-|Fcal| difference map (green mesh, 3.5σ) from a simulated annealing refinement omitting the R-12 RNA. (c). Schematic summary of Fab-RNA interactions on R-12 hairpin. Rectangles represent nucleobases and ovals represent CDR residues colored as in (d). (d). Molecular surface of the Fab-BRG variable domain in complex with the RNA. (e). Interaction between the 12mer RNA and the CDR residues. Orange spheres represent water molecules that mediate hydrogen bonds. Green dashes represent hydrogen bonds, and the red dash represents a salt bridge. Colors correspond to amino acid types (Tyr: yellow, Gly: green, Ser: red, Arg: blue, and others: magenta).
Figure 4
Figure 4
Interactions within the Fab-RNA binding interface. (a). Dimerization of the oligonucleotide observed in crystal lattice. The symmetry-related molecule is shown in pale color, with primes after its nucleotide numbers. (b–f) Close-up views of Fab-RNA interactions involving (b) CDR-L2, U2 and A3; (c) CDR-H3, U4, G5 and C12 (d) CDR-H3, C6, A7 and U8; (e) CDR-H2, A9 and G10; (f) CDR-H3, G10 and G11. Dashed lines with numbers indicating distances in Å: black – Fab-RNA hydrogen bonds, yellow – GC-pair hydrogen bonds, red – electrostatic, orange – hydrophobic. Residue color code matches Figure 3b.
Figure 5
Figure 5
R-12 RNA undergoes a shift in conformation upon Fab-BRG binding. (a). RNA oligonucleotides drawn in their putative hairpin conformations. All variants contain the AUAG tetraloop nucleotides were designed to differ in the number helical base pairs (b). EMSA assays (20% acrylamide) for RNA oligonucleotides containing 1, 2, 3 and 5 putative base pairs. The 2-bp hairpin migrates in a manner similar to 1-bp oligonucleotides but slower than the 3-bp and 5-bp oligonucleotide. (c). In-line probing analysis of 2-bp and 3-bp oligonucleotides. Input – control samples without probing buffer treatment (d). Quantitative analysis of the in-line probing results from (c). Positions 5, 6, and 11 react faster in the 2-bp oligonucleotide compared to 3-bp, whereas position 7, 8, 9, and 10 react to similar extent. Degradation counts from input are subtracted from each band in quantification. Data represent mean values ± s.d. from two experiments. (e). Effect of the number and identity of stem base-pairs on Fab-BRG binding (See Table 1 for more details).

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