Abstract
Spinach is an in vitro–selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.
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Acknowledgements
We are grateful to I.M. Steele for assistance in determining the structure of unbound DFHBI. We thank L. Zhang for the advice on structure and model building. We thank J.R. Fuller for refinement software support. We also thank F.C. Chou and R. Das for their aid with ERRASER software and K.N. Dyer, T.R. Sosnick and J.R. Hinshaw for the help with SAXS experiments. We thank members of the Piccirilli group, J.P. Staley and D.M.J. Lilley for helpful discussions and comments on the manuscript. The work is supported by US National Institutes of Health (NIH) grants R01-AI081987, NIH training grant T32GM008720 (to M.E.E.) and R01-GM102489 (to J.A.P.), NIH training grant T32GM007183 (to N.B.S.) and US National Institute of General Medical Sciences Medical Scientist National Research Service Award no. 5 T32GM07281 (to Y.K.). This work is based on research conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamline 24-ID-C&E, GM/CA beamline 23-ID-D and Advanced Light Source beamline 12.3.1 SIBYLS, all supported by USA Department of Energy.
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H.H. and J.A.P. designed the project; H.H. conducted most of the biochemical and biophysical assays and crystallography; N.B.S. and P.A.R. made essential contributions to crystallography; N.-S.L. synthesized DFHBI and analogs; S.A.S. and M.E.E. constructed and characterized truncation mutants; Y.K. developed the Fab BL3-6 chaperone; and J.A.P. provided overall project supervision. The manuscript was prepared by H.H., P.A.R. and J.A.P.
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Huang, H., Suslov, N., Li, NS. et al. A G-quadruplex–containing RNA activates fluorescence in a GFP-like fluorophore. Nat Chem Biol 10, 686–691 (2014). https://doi.org/10.1038/nchembio.1561
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DOI: https://doi.org/10.1038/nchembio.1561
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