A Multicolor Large Stokes Shift Fluorogen‐Activating RNA Aptamer with Cationic Chromophores

Abstract

Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4‐hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes‐shifted emission from several new green and red HBI analogues that are non‐fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functionalization at the C2 position of the imidazolone, this strategy yielded DMHBO+, which binds to the Chili aptamer with a low‐nanomolar KD. Because of its highly red‐shifted fluorescence emission at 592 nm, the Chili–DMHBO+ complex is an ideal fluorescence donor for Förster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET‐based analytical RNA systems.

Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase‐Modified Fluorogenic RNA Aptamers

“…Fluorogenic aptamers gained increasing interest as functional RNAs that light up conditionally fluorescent small molecules only when these are bound to the RNA . Recent developments include the families of Spinach, Broccoli, Corn and Chili aptamers for hydroxybenzylidene imidazolone (HBI)‐derived ligands, Mango variants for thiazole orange as well as the silicon rhodamine‐binding SiRA . Such in vitro‐selected RNA aptamers have found applications as genetically encodable tags for RNA imaging, as components of biosensors for metabolite detection, and as building blocks for responsive nucleic acid architectures .…”

“…Unifying these concepts, we introduce a new fluorescent nucleobase analogue–ligand FRET pair for studying RNA aptamer structure and folding. In particular, we investigated the Chili RNA aptamer, which exhibits large Stokes shift fluorescence emission of the cationic HBI chromophores DMHBI + and DMHBO + (Figure a,b) . Herein, we demonstrate that these ligands serve as supramolecular FRET acceptors of site‐specifically modified RNA aptamers that carry the fluorescent nucleobase analog 4‐cyanoindole (4CI) (Figure c).…”

“…An r4CI ‐modified Chili–HBI complex therefore constitutes an intrasupramolecular FRET pair, whose Förster radius ( R 0 ) depends on the context‐sensitive donor quantum yield in addition to the relative orientation of the FRET partners, which must be considered as static and non‐isotropic. We selected eight suitable incorporation sites for r4CI within the top and bottom stems, the tetraloop and the internal loop of the aptamer, based on previously reported Tb 3+ probing and mutagenesis experiments . The full‐length r4CI ‐labeled Chili aptamers were prepared from two synthetic 26 nt fragments by enzymatic ligation with T4 RNA ligase and characterized by ESI‐MS (Tables S1 and S2).…”

From fluorescent proteins to fluorogenic RNAs: Tools for imaging cellular macromolecules

“…When complexed with the compact aptamer “13‐2 min” that was originally isolated by Jaffrey and coworkers in the same selection experiment that yielded Spinach, three of these new fluorophores (DMHBI‐Imi, DMHBI + , and DMHBO + ; Fig. ) fluoresced green, yellow, and red, leading the authors to term the complexes Chili . In addition to providing access to a broad spectral range, Chili also has the largest Stokes shift reported for fluorogenic aptamers, ~130 nm for each of the DMHBI analogs.…”

Noncovalent Spin‐Labeling of DNA and RNA Triplexes

“…Aptamers are one class of nucleic acid binding motifs that have been used to bind fluorophores . A range of aptamers are available for this purpose . Optimized aptamers that bind brightly fluorescent dyes allow for intracellular tracking of RNA via super‐resolution, even for bacterial cells .…”