A homodimer interface without base pairs in an RNA mimic of red fluorescent protein

Abstract

Corn, a 28-nucleotide RNA, increases yellow fluorescence of its cognate ligand 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO) by >400-fold. Corn was selected in vitro to overcome limitations of other fluorogenic RNAs, particularly rapid photobleaching. We now report the Corn–DFHO co-crystal structure, discovering that the functional species is a quasisymmetric homodimer. Unusually, the dimer interface, in which six unpaired adenosines break overall two-fold symmetry, lacks any intermolecular base pairs. The homodimer encapsulates one DFHO at its interprotomer interface, sandwiching it with a G-quadruplex from each protomer. Corn and the green-fluorescent Spinach RNA are structurally unrelated. Their convergent use of G-quadruplexes underscores the usefulness of this motif for RNA-induced small-molecule fluorescence. The asymmetric dimer interface of Corn could provide a basis for the development of mutants that only fluoresce as heterodimers. Such variants would be analogous to Split GFP, and may be useful for analyzing RNA co-expression or association, or for designing self-assembling RNA nanostructures.

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Figure 1: Structure of Corn–DFHO complex.
Figure 2: Biophysical analysis of Corn–DFHO dimer.
Figure 3: Structure of Corn protomer.
Figure 4: Architecture and functional importance of the quasisymmetric DFHO binding site.
Figure 5: Comparison of chromophore binding and fluorescence activation by Spinach and Corn.

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Acknowledgements

We thank the staff at beamlines 5.0.1 and 5.0.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory, and beamline 24-ID-E of the Advanced Photon Source (APS), Argonne National Laboratory, for crystallographic data collection; the staff at APS beamline ID-12-B for SAXS; G. Piszczek (Biophysics Core, US National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH)) for analytical ultracentrifugation; D. Lee and R. Levine (NHLBI) for mass spectrometry; S. Bachas, M. Chen, C. Fagan, C. Jones, R. Trachman, M. Warner, and J. Zhang for discussions, and an anonymous referee for proposing the experiment in Supplementary Figure 3. This work was partly conducted at the ALS, on the Berkeley Center for Structural Biology Beamlines, and at the APS on the 24-ID-E (NE-CAT) and 12-ID-C beamlines, which are supported by the NIH. Use of ALS and APS was supported by the US Department of Energy. This work was supported in part by the NIH (R01 NS064516 S.R.J.), the NIH–Oxford–Cambridge Research Scholars Program (K.D.W.), and the intramural program of the NHLBI and NIH.

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A.R.F.-D. and S.R.J. conceived the project; W.S. and G.S.F. synthesized the chromophore and initial aptamers; K.D.W. designed and carried out crystallographic experiments; K.D.W. and L.S. performed biochemical and biophysical experiments, and K.D.W. and A.R.F.-D prepared the manuscript with input from all authors.

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Correspondence to Adrian R Ferré-D'Amaré.

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Competing interests

S.R.J. is the co-founder of Lucerna Technologies and has equity in this company. Lucerna has licensed commercialization of technology related to Spinach and other RNA -flourophore complexes.

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Supplementary Results, Supplementary Tables 1–5 and Supplementary Figures 1–8 (PDF 3211 kb)

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Warner, K., Sjekloća, L., Song, W. et al. A homodimer interface without base pairs in an RNA mimic of red fluorescent protein. Nat Chem Biol 13, 1195–1201 (2017). https://doi.org/10.1038/nchembio.2475

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