A Strategy for the Development of Small-Molecule-Based Sensors That Strongly Fluoresce When Bound to a Specific RNA

Abstract

We report a broadly applicable approach for the development of small-molecule-based RNA sensors. Our photoinduced electron transfer (PET) sensor consists of a fluorescein derivative as the fluorophore and two aniline derivatives as electron donors (quenchers). The isolation of electron-donor-binding RNA by in vitro selection (also known as SELEX) yielded an RNA aptamer that could increase the fluorescence intensity of the sensor by 13-fold. This result shows that RNA-electron-donor interactions can be used to develop modular RNA chemosensors.

Light-up properties of complexes between thiazole orange-small molecule conjugates and aptamers

“…Tsien and colleagues were first to report that malachite green in a complex with its RNA aptamer (10) has 2000-fold increase in the quantum yield (5). Sparano and Koide selected the light-up RNA aptamers for quencher modules in the engineered molecular quencher-fluorophore couples (4,6), while Sando's group reported the selection of DNA and RNA aptamers for derivatives of Hoechst 33258 (7,8). Most recently, Armitage and colleagues described the selection of the tightly binding RNA aptamer for an engineered cyanine dye (dimethylindole red) with 60-fold fluorescence enhancement (9).…”

Aptamer-Based Fluorescent Biosensors

“…For example, quencher-conjugated beads were used to screen out quencher-specific RNA aptamers which could separate a fluorophore-quencher pair, and/or lower the energy level of the quencher to reduce its FRET efficiency, upon binding thereby recovering the fluorescence [80,81]. Although µM concentrations of RNA aptamers could be detected and this interesting design may be incorporated into future SELEX of aptamers, potential applications of this strategy are limited since the target has to be an effective fluorescence quencher.…”

New Ideas for in Vivo Detection of RNA

“…As a group, their emission maxima cover much of the visible spectrum and extend into the near infrared. Another recently developed approach for creating light-up dyes is to start with a dye that emits when free in solution and to covalently attach electron donor groups that quench its emission by a photo-induced electron transfer (PET) process (Sparano & Koide, 2005;Sparano & Koide, 2007). Aptamers that bind exclusively to the quencher moieties are then selected.…”

“…In case of MG, phenyl ring (PhMG) mentioned in text is circled. D. Schematic representation of a strategy use to obtain a new " light-up" pair (Sparano & Koide, 2005). Dichlorofluorescein (DCF, shown in green) is attached to two quencher molecules (shown in black).…”