A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination

Abstract

RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab–ligase complex at 3.1-Å resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs′ high molecular weight, large surface area and phasing power.

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Figure 1: Selection of class I ligase–binding Fabs from the YSGR Fab superlibrary.
Figure 2: Affinity maturation of class I ligase–binding Fab-BL3 by error-prone PCR.
Figure 3: Crystal structure of the Fab BL3-6–ligase complex.
Figure 4: Details of Fab-P5 loop interactions.
Figure 5: Fab–ligase crystal packing.
Figure 6: Analysis of the Fab–ligase P5 epitope.

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Acknowledgements

We thank members of the Piccirilli, Koide and Kossiakoff laboratories for assistance with phage display and for insightful comments; D. Lilley and T. Wilson for helpful discussions regarding the VS ribozyme; and V. Torbeev, I. Dementieva, P. Rice and V. Tereshko for assistance with crystallography. This work was funded by Howard Hughes Medical Institute (J.A.P.), US National Institute of General Medical Sciences Medical Scientist National Research Service Award no. 5 T32 GM07281 (Y.K.) and US National Institutes of Health grants R01-GM72688 and U54-GM74946 (to S.K. and A.A.K.) and R01-GM61835 (to D.P.B.). Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research program (grant 085P1000817).

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All authors designed research; Y.K., E.M.D., D.M.S. and N.B.S. performed experiments; Y.K., E.M.D., D.M.S., S.K., A.A.K. and J.A.P. analyzed data; Y.K., E.M.D., D.M.S., N.B.S., D.P.B., S.K., A.A.K. and J.A.P wrote the paper.

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Correspondence to Anthony A Kossiakoff or Joseph A Piccirilli.

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The authors declare no competing financial interests.

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Supplementary Methods, Supplementary Note and Supplementary Figures 1–6 (PDF 5448 kb)

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Koldobskaya, Y., Duguid, E., Shechner, D. et al. A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination. Nat Struct Mol Biol 18, 100–106 (2011). https://doi.org/10.1038/nsmb.1945

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