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A bright FIT-PNA hybridization probe for the hybridization state specific analysis of a C → U RNA edit via FRET in a binary system.

Fang, Ge-Min and Chamiolo, Jasmine and Kankowski, Svenja and Hövelmann, Felix and Friedrich, Dhana and Loewer, Alexander and Meier, Jochen C. and Seitz, Oliver (2018):
A bright FIT-PNA hybridization probe for the hybridization state specific analysis of a C → U RNA edit via FRET in a binary system.
In: Chemical science, pp. 4794-4800, 9, (21), ISSN 2041-6520, [Article]

Abstract

Oligonucleotide probes that show enhanced fluorescence upon nucleic acid hybridization enable the detection and visualization of specific mRNA molecules, and . A challenging problem is the analysis of single nucleotide alterations that occur, for example, when cellular mRNA is subject to C → U editing. Given the length required for uniqueness of the targeted segment, the commonly used probes do not provide the level of sequence specificity needed to discriminate single base mismatched hybridization. Herein we introduce a binary probe system based on fluorescence resonance energy transfer (FRET) that distinguishes three possible states (i) absence of target, (ii) presence of edited (matched) and (iii) unedited (single base mismatched) target. To address the shortcomings of read-out FRET, we designed donor probes that avoid bleed through into the acceptor channel and nevertheless provide a high intensity of FRET signaling. We show the combined use of thiazole orange (TO) and an oxazolopyridine analogue (JO), linked as base surrogates in modified PNA FIT-probes that serve as FRET donor for a second, near-infrared (NIR)-labeled strand. In absence of target, donor emission is low and FRET cannot occur of the lacking co-alignment of probes. Hybridization of the TO/JO-PNA FIT-probe with the (unedited RNA) target leads to high brightness of emission at 540 nm. Co-alignment of the NIR-acceptor strand ensues from recognition of edited RNA inducing emission at 690 nm. We show imaging of mRNA in fixed and live cells and discuss the homogeneous detection and intracellular imaging of a single nucleotide mRNA edit used by nature to post-transcriptionally modify the function of the Glycine Receptor (GlyR).

Item Type: Article
Erschienen: 2018
Creators: Fang, Ge-Min and Chamiolo, Jasmine and Kankowski, Svenja and Hövelmann, Felix and Friedrich, Dhana and Loewer, Alexander and Meier, Jochen C. and Seitz, Oliver
Title: A bright FIT-PNA hybridization probe for the hybridization state specific analysis of a C → U RNA edit via FRET in a binary system.
Language: English
Abstract:

Oligonucleotide probes that show enhanced fluorescence upon nucleic acid hybridization enable the detection and visualization of specific mRNA molecules, and . A challenging problem is the analysis of single nucleotide alterations that occur, for example, when cellular mRNA is subject to C → U editing. Given the length required for uniqueness of the targeted segment, the commonly used probes do not provide the level of sequence specificity needed to discriminate single base mismatched hybridization. Herein we introduce a binary probe system based on fluorescence resonance energy transfer (FRET) that distinguishes three possible states (i) absence of target, (ii) presence of edited (matched) and (iii) unedited (single base mismatched) target. To address the shortcomings of read-out FRET, we designed donor probes that avoid bleed through into the acceptor channel and nevertheless provide a high intensity of FRET signaling. We show the combined use of thiazole orange (TO) and an oxazolopyridine analogue (JO), linked as base surrogates in modified PNA FIT-probes that serve as FRET donor for a second, near-infrared (NIR)-labeled strand. In absence of target, donor emission is low and FRET cannot occur of the lacking co-alignment of probes. Hybridization of the TO/JO-PNA FIT-probe with the (unedited RNA) target leads to high brightness of emission at 540 nm. Co-alignment of the NIR-acceptor strand ensues from recognition of edited RNA inducing emission at 690 nm. We show imaging of mRNA in fixed and live cells and discuss the homogeneous detection and intracellular imaging of a single nucleotide mRNA edit used by nature to post-transcriptionally modify the function of the Glycine Receptor (GlyR).

Journal or Publication Title: Chemical science
Volume: 9
Number: 21
Divisions: 10 Department of Biology
10 Department of Biology > Systems Biology of the Stress Response
Date Deposited: 26 Jun 2018 08:48
Identification Number: pmid:29910930
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