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Structural basis for activity of highly efficient RNA mimics of green fluorescent protein

Nature Structural & Molecular Biology volume 21, pages 658663 (2014) | Download Citation

Abstract

GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro–evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without resorting to large RNA-binding protein–GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.

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Acknowledgements

We thank the staff at beamlines 5.0.2 of the Advanced Light Source (ALS), 24-ID-C of the Advanced Photon Source (APS) and 11-1 of the Stanford Synchrotron Radiation Lightsource (SSRL) for crystallographic data collection; G. Piszczek (US National Heart, Lung and Blood Institute, NHLBI) for fluorescence spectroscopy; X. Fang (US National Cancer Institute) and the staff of APS 12-ID-C for SAXS; D.-Y. Lee (NHLBI) for MS; X. Wu (NHLBI) for fluorescence microscopy; N. Tjandra for NMR; J. Grimmett and T. Darling for MRC Laboratory of Molecular Biology computer-cluster support; and N. Baird, P. Emsley, C. Jones, F. Long, G. Murshudov, R. Nicholls, K. Perry, M. Lau, A. Roll-Mecak, M. Warner, K. Weeks and J. Zhang for discussions. This work was partly conducted at the ALS on the Berkeley Center for Structural Biology beamlines, at the APS on the 24-ID-C (NE-CAT) and 12-ID-C beamlines and at SSRL, which are all supported by the US National Institutes of Health (NIH, GM103403 and GM103393 to APS and SSRL, respectively). Use of ALS, APS and SSRL was supported by the US Department of Energy. This work was supported in part by the NIH (R01 NS010249 to S.R.J. and F32 GM106683 to R.L.S.), the European Union FP7 Marie-Curie IEF program (A.T.), the NIH-Oxford-Cambridge Research Scholars Program (K.D.W. and M.C.C.) and the intramural program of the NHLBI, NIH.

Author information

Affiliations

  1. Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA.

    • Katherine Deigan Warner
    • , Michael C Chen
    •  & Adrian R Ferré-D'Amaré
  2. Department of Pharmacology, Weill-Cornell Medical College, Cornell University, New York, New York, USA.

    • Wenjiao Song
    • , Rita L Strack
    •  & Samie R Jaffrey
  3. Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge, UK.

    • Andrea Thorn

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Contributions

K.D.W. and A.R.F.-D. designed experiments; W.S., R.L.S. and S.R.J. synthesized chromophores and some aptamers; K.D.W. carried out biochemistry, crystallization and SAXS; K.D.W. and A.R.F.-D. collected diffraction data; K.D.W., A.T. and A.R.F.-D. reduced data; A.T. solved the heavy atom substructure and calculated initial phases; K.D.W. built the crystallographic model, and K.D.W. and A.T. refined it; M.C.C. performed NMR; and A.R.F.-D. and K.D.W. wrote the manuscript with help from M.C.C., A.T. and S.R.J., and all authors reviewed it.

Competing interests

S.R.J. and R.L.S. are authors of a patent application (provisional patent USPTO no. 61/874,819) related to RNA–fluorophore complexes described in this paper. The other authors declare no competing financial interests.

Corresponding author

Correspondence to Adrian R Ferré-D'Amaré.

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https://doi.org/10.1038/nsmb.2865

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