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

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.

Figure 1

Structure of the Spinach-DFHBI complex. (a) Chemical structures^, of cis-DFHBI and cis-DBrHBI. (b) Sequence and secondary structure of Spinach-DFHBI. Thin lines denote chain connectivity; Leontis-Westhof symbols, non-canonical base pairs. Numbering scheme for Spinach1.2 (ref. 8) is used throughout. (c) Cartoon representation, color-coded as in b. Purple spheres represent K⁺.

Figure 2

The Spinach chromophore binding site contains a G-quadruplex. (a) Green mesh depicts a portion of the |F_o|-|F_c| electron density map, calculated prior to addition of DFHBI to the crystallographic model, contoured at 4 s.d. (b) Water and K⁺ bind the difluorohydroxyphenyl ring of DFHBI. (c) Connectivity and stereochemistry of the Spinach G-quadruplex. Light and dark shades denote anti and syn conformations, respectively. Empty and filled circles denote C3'-endo and C2'-endo puckers, respectively (G68 is O4'-endo). (d) The two G-quartets and cation M_A. Green and black dashes represent cation coordination and hydrogen bonds, respectively. (e) The mixed tetrad, lower G-quartet, and cation M_B.

Figure 3

Comparison of GFP and Spinach. (a) Molecular surface of Spinach-DFHBI showing the imidazolone ring and the 'gateway' A69. (b) Cartoon and molecular surface of GFP. The chromophore is green. (c) Interior molecular surface surrounding the Spinach chromophore in gray. (d) Surface of the chromophore binding pocket of enhanced GFP (PDB: 4EUL; ref. 39) in gray.

Figure 4

G-quadruplexes in Spinach and other fluorogenic RNAs. (a) Fluorescence of mutant Spinach RNAs in excess DFHBI, normalized to Spinach fluorescence. Error bars represent standard errors of the mean. N.D., not detected. (b-d) Imino region of the proton NMR spectra of Spinach, Baby Spinach and '13-2 min' RNAs, in the presence and absence of DFHBI. (e) Fluorescence emission spectra of Spinach, Baby Spinach, and 13–2 min bound to DFHBI, normalized by peak absorbance. The spectra of Spinach and Baby Spinach superimpose exactly.