Dataset Information

Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase-Modified Fluorogenic RNA Aptamers.

ABSTRACT: RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence-based strategies reveal information on structure and dynamics of RNA aptamers. Herein, we report the incorporation of the universal emissive nucleobase analog 4-cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to the bound ligands DMHBI+ or DMHBO+ . The photophysical properties of the new nucleobase-ligand-FRET pair revealed structural restraints for the overall RNA aptamer organization and identified nucleotide positions suitable for FRET-based readout of ligand binding. This strategy is generally suitable for binding-site mapping and may also be applied for responsive aptamer devices.

SUBMITTER: Steinmetzger C

PROVIDER: S-EPMC7187157 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase-Modified Fluorogenic RNA Aptamers.

Steinmetzger Christian C Bäuerlein Carmen C Höbartner Claudia C

Angewandte Chemie (International ed. in English) 20200306 17

RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence-based strategies reveal information on structure and dynamics of RNA aptamers. Herein, we report the incorporation of the universal emissive nucleobase analog 4-cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to the bound ligands DMHBI<sup>+</sup> or DMHBO<sup>+</sup> . The photophysical properties of the new nucleobase-ligand-FRE ...[more]

PMID: 32052536

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Project description:It has been shown that fluorescence resonance energy transfer (FRET)-based PCR, including the TaqMan assay and molecular beacons, has potential for rapid detection of pathogens. In these promising real-time detection assays a single internal oligonucleotide probe labeled on both the 5' (reporter) and 3' (quencher) ends is used for selective generation of fluorescence. In this paper, we describe the use of a previously reported novel probe design for FRET-based PCR detection of Listeria monocytogenes in pure culture and in a model food commodity. In the assay described here an asymmetric probe set is used; this probe set consists of a long 5' fluorescein-labeled reporter probe and a short, complementary 3' DABCYL-labeled quencher oligonucleotide, which are used in a 5' nuclease amplification and detection assay. By using the listeriolysin O (hly) and p60 (iap) genes as amplification targets, the performance of two primer-probe sets in amplification and subsequent detection of target DNA was evaluated. In studies performed with pure cultures of L. monocytogenes, the PCR profiles indicated that the relative change in fluorescence intensity was correlated with both the initial number of cells and the accumulation of specific amplicons for both hly and iap gene fragments. Experiments were also done to determine the applicability of the method to the detection of L. monocytogenes by targeting hly DNA and its short-lived mRNA product in a model food commodity. Twenty-five-milliliter samples of reconstituted nonfat dry milk (NFDM) were seeded with L. monocytogenes and processed to concentrate the bacteria by centrifugation, and this was followed by nucleic acid extraction and amplification with hly-specific primers. Endpoint detection of PCR and reverse transcription-PCR amplicons could be achieved at inoculum levels of 10(3) and 10(4) CFU of L. monocytogenes/25 ml of NFDM, respectively. This study demonstrated that this asymmetric FRET-based amplification and detection protocol provides an alternative approach for endpoint detection of nucleic acid amplification products as applied to detection of pathogens in a model food.

Dataset Information

Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase-Modified Fluorogenic RNA Aptamers.

Publications

Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase-Modified Fluorogenic RNA Aptamers.

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