title: Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection.
abstract: A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics.
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Jieqiong
authors.initials: J
authors.lastname: Qiu
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Adam
authors.initials: A
authors.lastname: Wilson
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez University, Suez, 43721, Egypt.
authors.forename: Afaf H
authors.initials: AH
authors.lastname: El-Sagheer
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK tom.brown@chem.ox.ac.uk.
authors.forename: Tom
authors.initials: T
authors.lastname: Brown
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D052160:Benzothiazoles / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D017930:Genes, Reporter; D007364:Intercalating Agents / Q000737:chemistry / Q000378:metabolism*; D008745:Methylation; D009154:Mutation / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000235:genetics; D011804:Quinolines / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000032:analysis*; D013050:Spectrometry, Fluorescence
chemical_list: D052160:Benzothiazoles; D015342:DNA Probes; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D011804:Quinolines; C051000:thiazole orange; D012313:RNA; D004247:DNA; C005465:benzothiazole