Imaging of RNA in Bacteria with Self-Ligating Quenched Probes

Abstract

We report on the application of a new class of oligonucleotide reporter probes, QUAL probes, that "light up" when a nucleophilic phosphorothioate probe binds adjacent to a dabsyl-quenched probe. These self-ligating DNA probes were used for sequence-specific detection of 16S rRNA in Escherichia coli cells. Strong fluorescence was observed only when the phosphorothioate and quenched dabsyl probes bind side-by-side on a 16S rRNA target. The results demonstrate the use of QUAL probes to detect specific RNA sequences in bacterial cells without enzymes and without washing steps.

New, stronger nucleophiles for nucleic acid-templated chemistry: Synthesis and application in fluorescence detection of cellular RNA

“…[14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] Our own research in nucleic acid templated chemistry has focused recently on detection of RNAs directly in cells. 20,21,32,33 This application is potentially important for rapid identification of pathogenic bacteria and for identification of disease-related messenger RNAs in human cells. We have previously described nucleophilic chemistry for templated ligations of modified DNA probes, 17,19 and have developed quencher-based leaving groups that yield a fluorescent signal upon reaction.…”

“…We have previously described nucleophilic chemistry for templated ligations of modified DNA probes, 17,19 and have developed quencher-based leaving groups that yield a fluorescent signal upon reaction. 32 We also developed a product destabilization strategy for engendering turnover of reactive probes on a template, thus yielding amplification of signal. 21 With DNA or RNA detection as a focus, the large majority of nucleic acid-templated reactions have involved chemical joining of a pair of probes.…”

Subnanomolar Detection of Oligonucleotides through Templated Fluorogenic Reaction in Hydrogels: Controlling Diffusion to Improve Sensitivity

“…In such cases, the fluorescence intensity emitted by the product of the OTR is directly proportional to the amount of NA target present, whilst only very low levels of background fluorescence can be detected in the absence of the template (leading to a high signal‐to‐noise ratio or S/N). Representative examples include the quenched auto‐ligation (QUAL)4b, 5a,5b strategy in which an OTR causes the release of a quencher molecule, resulting in the restoration of the intrinsic fluorescence of an otherwise quenched nearby fluorophore. Another common strategy uses an oligonucleotide template to catalyze the formation of a fluorescent dye from two non‐ or weakly fluorescent precursors 4c, 6.…”

Flow cytometric detection of specific RNAs in native human cells with quenched autoligating FRET probes

“…However, a small number of molecular approaches are under development (3,4,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Prominent among these approaches are quenched probe strategies, including molecular beacon (MB) probes (19,(22)(23)(24) and quenched autoligating (QUAL) probes (25)(26)(27)(28). Quenched probe strategies offer the advantage of decreased background signal in the absence of the RNA target (29).…”

“…Because of the short length of these QUAL probes before autoligation, their binding to mismatched targets is weak, slowing ligation and yielding high selectivity for single-nucleotide differences (25). By this approach, high-copy-number 16S rRNA sequences in living bacterial cells were imaged with single-nucleotide discrimination (26). However, background signals were significant, and no prior studies exist on the application of QUAL probes in detection of less-abundant messenger RNAs.…”