Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine

Mateusz Mieczkowski,Irene Bessi,Claudia Höbartner,Marco Holzapfel,Christian Steinmetzger,Christoph Lambert,Ann-Kathrin Lenz,Vladimir Pena,Alexander Schmiedel

doi:10.1038/s41467-021-23932-0

Abstract

Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural and mechanistic basis of fluorescence activation by crystallography and time-resolved optical spectroscopy. Two co-crystal structures of the Chili RNA with positively charged DMHBO+ and DMHBI+ ligands revealed a G-quadruplex and a trans-sugar-sugar edge G:G base pair that immobilize the ligand by π-π stacking. A Watson-Crick G:C base pair in the fluorophore binding site establishes a short hydrogen bond between the N7 of guanine and the phenolic OH of the ligand. Ultrafast excited state proton transfer (ESPT) from the neutral chromophore to the RNA was found with a time constant of 130 fs and revealed the mode of action of the large Stokes shift fluorogenic RNA aptamer.

Highlights

Fluorogenic RNA aptamers are synthetic functional RNAs that bind and activate conditional fluorophores
Functional characterization of the Chili RNA has indicated that a G-quadruplex is likely involved in the RNA–ligand interaction[8,18], consistent with earlier hints obtained for the 13-2 aptamer with DFHBI20
Overall structure of the Chili RNA aptamer bound to DMHBO+ and dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI)+

Summary

Introduction

Fluorogenic RNA aptamers are synthetic functional RNAs that bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. The class of Spinach, Broccoli, Corn, and Chili are RNA mimics of green fluorescent proteins (GFP) that bind and activate derivatives of 4-hydroxybenzylidene imidazolone (HBI), a smallmolecule analog of the tripeptide chromophore in GFP13. 52 nt Chili RNA preferentially binds and activates the positively charged chromophores DMHBI+ and DMHBO+ (Fig. 1a, b)[8,18] and mimics large Stokes shift fluorescent proteins[19]. The structure suggests a model for the excited state proton transfer (ESPT) that is supported by time-resolved spectroscopy These insights enabled structure-guided miniaturization of the Chili aptamer and will aid future engineering of fluorogenic modules for sensors and imaging applications

Methods

Results

Conclusion