Multiscale Simulations on Spectral Tuning and the Photoisomerization Mechanism in Fluorescent RNA Spinach.

Fluorescent RNA aptamer Spinach can bind and activate a green fluorescent protein (GFP)-like chromophore (an anionic DFHBDI chromophore) displaying green fluorescence. Spectroscopic properties, spectral tuning, and the photoisomerization mechanism in the Spinach-DFHBDI complex have been investigated by high-level QM and hybrid ONIOM(QM:AMBER) methods (QM method: (TD)DFT, SF-BHHLYP, SAC-CI, LT-DF-LCC2, CASSCF, or MS-CASPT2), as well as classical molecular dynamics (MD) simulations. First, our benchmark calculations have shown that TD-DFT and spin-flip (SF) TD-DFT (SF-BHHLYP) failed to give a satisfactory description of absorption and emission of the anionic DFHBDI chromophore. Comparatively, SAC-CI, LT-DF-LCC2, and MS-CASPT2 can give more reliable transition energies and are mainly used to further study the spectra of the anionic DFHBDI chromophore in Spinach. The RNA environmental effects on the spectral tuning and the photoisomerization mechanism have been elucidated. Our simulations show that interactions of the anionic cis-DFHBDI chromophore with two G-quadruplexes as well as a UAU base triple suppress photoisomerization of DFHBDI. In addition, strong hydrogen bonds between the anionic cis-DFHBDI chromophore and nearby nucleotides facilitate its binding to Spinach and further inhibit the cis-trans photoisomerization of DFHBDI. Solvent molecules, ions, and loss of key hydrogen bonds with nearby nucleotides could induce dissociation of the anionic trans-DFHBDI chromophore from the binding site. These results provide new insights into fluorescent RNA Spinach and may help rational design of other fluorescent RNAs.