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

Nature Structural & Molecular Biology volume 21, pages 658–663 (2014)Cite this article

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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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Figure 1: Structure of the Spinach–DFHBI complex.
Figure 2: The Spinach chromophore-binding site contains a G-quadruplex.
Figure 3: Comparison of GFP and Spinach.
Figure 4: G-quadruplexes in Spinach and other fluorogenic 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.

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Authors and 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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  1. Katherine Deigan Warner
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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.

Corresponding author

Correspondence to Adrian R Ferré-D'Amaré.

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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.

Integrated supplementary information

Supplementary Figure 1 Crystallographic and SAXS characterization of Spinach.

(a) Comparison of the fluorescence spectra of unimolecular and split (crystallization) Spinach1.2 RNA constructs (Supplementary Table 2) bound to DFHBI. (b) Portion of the density-modified SAD electron density map corresponding to the two G-quartets contoured at 1 s.d. (gray mesh). Green mesh depicts the anomalous difference Fourier synthesis contoured at 4 s.d. Both maps calculated with Crystal I data (Table 1). (c) Portion of the anomalous difference Fourier synthesis (magenta mesh) calculated with data from the residue 18 5-iodouridine Split Spinach-DFHBI co-crystal (Crystal III, Table 1) contoured at 4 s.d., superimposed on the final refined model for Crystal I. (d) Portion of the anomalous difference Fourier synthesis (magenta mesh) calculated with data from the DBrHBI co-crystal (Crystal IV, Table 1) contoured at 4 s.d., superimposed on the DFHBI from the molecular replacement model. (e) P(r) functions for free and DFHBI-bound Spinach RNA. (f) Comparison of the scattering profile back-calculated from the model of DFHBI-bound Spinach (Crystal I) and the experimental solution X-ray scattering of DFHBI-bound Spinach RNA (RNA 12, Supplementary Table 2). (g) Kratky analysis of experimental free and DFHBI-bound Spinach RNA SAXS data.

Supplementary Figure 2 Secondary structure of Spinach.

(a) Secondary structure of Spinach previously proposed6 based on computational structure prediction. Nucleotides are colored to match Fig. 1b. Base-pairing interactions not observed in the Spinach-DFHBI co-crystal structure are denoted by dashed red lines. (b) The secondary structure adopted by Spinach in complex with DFHBI is compatible with the Spinach1, Spinach1.2 and Spinach2 sequence variants6,8. Red squares indicate sequence differences from Spinach1.2.

Supplementary Figure 3 Metal-ion interactions with Spinach.

(a) In the Crystal I crystallographic model, the difluorohydroxyphenyl ring of DFHBI is coordinated by four waters as well as K+ (Mc), and the 2′-OH of G26. An additional K+ (MD) and water molecules are also present in the cation-binding site. (b) In the Crystal II crystallographic model, the difluorohydroxyphenyl ring of DFHBI is coordinated by a Mg2+ (Mc) and the 2′-OH of G26. Two ordered water molecules are also present in the cation-binding site. (c) Spinach-bound DFHBI is partially accessible to bulk solvent. Molecular surface of the complex. View of the DFHBI binding site is as in a and Fig. 2b. (d) View is as in Fig. 2a. (e) Histogram of fluorescence of 1 μM of Spinach RNA and 10 μM DFHBI in the presence of various cations, normalized to Spinach fluorescence (RNA 3 and 4, Supplementary Table 2) in the 0.125 M KCl, 5 mM MgCl2 condition. Error bars represent s.e.m. ND, not determined. (f) Fluorescence emission spectra in the presence of various cations, normalized to Spinach fluorescence (RNA 3 and 4) in the 0.125 M KCl, 5 mM MgCl2 condition.

Supplementary Figure 4 The Spinach G-quadruplex tapers gradually to the canonical A-form antiparallel duplex P2.

(a) C1' to C1' distances (measured diagonally for the tetrads) are shown. (b) View as in a, but rotated 180° along the vertical axis.

Supplementary Figure 5 Structural requirements for DFHBI recognition by Spinach.

(a) View of the DFHBI binding site with cis-DFHBI bound, with metal ions and waters removed, from the final refined Crystal I structure. (b) View of the DFHBI bindings site modeled with trans-DFHBI comparison with a. The trans-DFHBI exhibits steric clashes and fewer hydrogen bonds than cis-DFHBI. (c) Alternate orientation of trans-DFHBI modeled for comparison, also showing fewer favorable interactions than a. (d) Hypothetical secondary structure of Baby Spinach. Nucleotides are colored to match Fig. 1b.

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Supplementary Figures 1–5 and Supplementary Tables 1 and 2 (PDF 7136 kb)

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Warner, K., Chen, M., Song, W. et al. Structural basis for activity of highly efficient RNA mimics of green fluorescent protein. Nat Struct Mol Biol 21, 658–663 (2014). https://doi.org/10.1038/nsmb.2865

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