Low‐Noise Stemless PNA Beacons for Sensitive DNA and RNA Detection

Socher, Elke; Bethge, Lucas; Knoll, Andrea; Jungnick, Nadine; Herrmann, Andreas; Seitz, Oliver

doi:10.1002/anie.200803549

Cited by 105 publications

(70 citation statements)

References 40 publications

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“…An extended applicability to sequence contexts has been reported for the dual-labeled TO-containing stem-less PNA beacons. 36 Whether such probes enable live cell RNA imaging remains to be evaluated. The use of differently colored PNA FIT-probes in this feasibility study revealed significant differences in the expression pattern of influenza H1N1 mRNAs coding for neuraminidase (NA) or matrix protein 1 (M1).…”

Section: ■ Discussionmentioning

confidence: 99%

PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells

et al. 2012

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Fluorogenic hybridization probes that allow RNA imaging provide information as to how the synthesis and transport of particular RNA molecules is orchestrated in living cells. In this study, we explored the peptide nucleic acid (PNA)-based FIT-probes in the simultaneous imaging of two different viral mRNA molecules expressed during the replication cycle of the H1N1 influenza A virus. PNA FIT-probes are non-nucleotidic, nonstructured probes and contain a single asymmetric cyanine dye which serves as a fluorescent base surrogate. The fluorochrome acts as a local intercalator probe and reports hybridization of target DNA/RNA by enhancement of fluorescence. Though multiplexed hybridization probes are expected to facilitate the analysis of RNA expression, there are no previous reports on the dual color imaging of two different viral mRNA targets. In this work, we developed a set of two differently colored PNA FIT-probes that allow the spectrally resolved imaging of mRNA coding for neuraminidase (NA) and matrix protein 1 (M1); proteins which execute distinct functions during the replication of the influenza A virus. The probes are characterized by a wide range of applicable hybridization temperatures. The same probe sequence enabled live-cell RNA imaging (at 37 °C) as well as real-time PCR measurements (at 60 °C annealing temperature). This facilitated a comprehensive analysis of RNA expression by quantitative (qPCR) and qualitative (imaging) means. Confocal laser scanning microscopy showed that the viral-RNA specific PNA FIT-probes neither stained noninfected cells nor cells infected by a control virus. The joint use of differently colored PNA FIT-probes in this feasibility study revealed significant differences in the expression pattern of influenza H1N1 mRNAs coding for NA or M1. These experiments provide evidence for the usefulness of PNA FIT-probes in investigations on the temporal and spatial progression of mRNA synthesis in living cells for two mRNA species.

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Section: ■ Discussionmentioning

confidence: 99%

PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells

et al. 2012

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“…The stemless PNA beacon was also used to detect the TAR loop from the HIV genome within an in vitro transcribed 650-nucleotide long TAR-CAT fusion, and up to 24-fold increase in signal was found. [95] Seitz also used PNA based hairpin peptide beacons in which the specific loop region was recognised by a protein target, which opens the hairpin structure increasing the fluorescence. Two targets were chosen: Src kinase and aspartic acid protease, rennin, which showed an 8-11-fold increase in fluorescence for the target proteins; this demonstrates the possibility for the use of hairpin peptide beacons in the detection of a broad range of proteins ( Figure 5).…”

Section: Bioanalytical and Diagnostic Applicationsmentioning

confidence: 99%

Chemical Modification of Oligonucleotides for Therapeutic, Bioanalytical and other Applications

2009

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Although known for more than a century, bromine trifluoride is not a common reagent in day to day research work in organic chemistry. Its tendency to react exothermically with solvents containing Lewis base elements such as water, acetone or ethers distanced it from the mind of many. Still, under the proper conditions, it can perform quite a few selective reactions which are difficult to achieve by other reagents. We discuss in this review reactions which have been published in the last 30 years. They consist of fluorinating heteroatoms, substituting carbon‐halogen bonds with carbon‐fluorine bonds, syntheses of anesthetics, construction of the trifluoromethyl (CF3) and the difluoromethylene (CF2) groups, aromatic brominations and more.

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“…Assignment of the H7, H8, H11, and H12 protons of U MBF were confirmed by the 2D DQF-COSY and NOESY spectra. Assignments of H2 protons of adenine and of H6, H8, and H1' protons of canonical deoxynucleotides and H7, H8, H11, and H12 proton of U MBF were confirmed by the 1 H, 13 C HSQC spectrum. Assignments of the methyl group of thymine and H2' and H2'' protons of deoxynucleotides were confirmed by the 2D TOCSY and NOESY spectra.…”

Section: Methodsmentioning

confidence: 80%

Controlling the Fluorescence of Benzofuran‐Modified Uracil Residues in Oligonucleotides by Triple‐Helix Formation

et al. 2014

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We developed fluorescent turn-on probes containing a fluorescent nucleoside, 5-(benzofuran-2-yl)deoxyuridine (dU(BF)) or 5-(3-methylbenzofuran-2-yl)deoxyuridine (dU(MBF)), for the detection of single-stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dU(MBF) achieved superior fluorescence enhancement than that containing dU(BF). NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex formation partly as a consequence of a conformational change at the bond between the 3-methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3-methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex-induced fluorescence enhancement of the oligonucleotide probe containing dU(MBF).

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Low‐Noise Stemless PNA Beacons for Sensitive DNA and RNA Detection

Cited by 105 publications

References 40 publications

PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells

PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells

Chemical Modification of Oligonucleotides for Therapeutic, Bioanalytical and other Applications

Controlling the Fluorescence of Benzofuran‐Modified Uracil Residues in Oligonucleotides by Triple‐Helix Formation

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