Fluorescent Sensors for Specific RNA:  A General Paradigm Using Chemistry and Combinatorial Biology

Abstract

Here, we describe a new paradigm for the development of small molecule-based RNA sensors. We prepared a series of potential PET (photoinduced electron transfer) sensors on the basis of 2',7'-dichlorofluorescein (DCF) fluorophore conjugated with two aniline derivatives as electron donors (quenchers). NMR and fluorescent spectroscopic analyses of these DCF derivatives revealed the correlation between the conformations, the PET, and the fluorescent intensities of these DCF derivatives, enabling us to select a sensor candidate. RNA aptamers were raised against the aniline-based quencher via in vitro selection (SELEX). One of these aptamers enhanced the fluorescence intensity of the DCF-aniline conjugate in a concentration-dependent manner. To demonstrate the power and generality of this approach, additional in vitro selection was performed and aptamers from this selection were found to have similar activities. These results show that one can develop fluorescence-inducing reporter RNA and morph it into remotely related sequences without prior structural insight into RNA-ligand binding.

Aptamer-based molecular imaging

“…Then an aptamer can be developed to either bind the whole dye (ASR7) or just the quencher (MPP in DCF-MPP or BHQ1 in fluorophore-BHQ1) (Sparano and Koide, 2007;Murata et al, 2011), resulting in fluorescence enhancement of 7-100 times.…”

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.…”

“…An RNA aptamer for the MPP quencher moiety was obtained by SELEX and was indeed found to enhance DCF fluorescence in the DCF/ MPP conjugate in a concentration-dependent manner. As the authors pointed out, however, this particular DCF-MPP/ aptamer system is unsuitable for in vivo applications because the fluorescence enhancement is only about 13-fold at 100 μM aptamer concentration (Sparano & Koide, 2007). A novel class of light-up probes for RNA detection was developed by Stojanovic's group by conjugating nonspecific fluorogenic intercalating dyes such as thiazole orange (TO) to small molecules such as GMP and AMP, for which RNA aptamers have previously been obtained (Pei et al, 2009).…”

Light‐Up Hoechst–DNA Aptamer Pair: Generation of an Aptamer‐Selective Fluorophore from a Conventional DNA‐Staining Dye

“…To this end, Sparano and Koide recently reported on a fluorophore-quencher photoinduced electron transfer (PET) system that exhibits fluorescence enhancement on binding to an RNA aptamer, selected in vitro, that perturbs the PET process. [10] Here, we show a potential approach for generating a bioorthogonal light-up fluorophore-aptamer pair from a single microenvironment-sensitive fluorophore. Our method allows for the conversion of a conventional double-stranded DNAstaining dye into an aptamer-selective light-up fluorophore, which can be applied for nucleic acid sensing with use of an unmodified DNA as a probe.…”