Journal
CHEMBIOCHEM
Volume 6, Issue 1, Pages 69-77Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200400260
Keywords
fluorescence spectroscopy; fluorescent probes; peptide nucleic acids; single-nucleotide polymorphisms; solid-phase synthesis
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Fluorescent base analogues in DNA are versatile probes of nucleic acid-nucleic acid and nucleic acid-protein interactions. New peptide nucleic acid (PNA) based probes are described in which the intercalator dye thiazole orange (TO) serves as a base surrogate. The investigation of six TO derivatives revealed that the linker length and the conjugation site decided whether a base surrogate conveys sequence-selective DNA binding and whether fluorescence is increased or decreased upon single-mismatched hybridization. One TO derivative conferred universal PNA-DNA base pairing while maintaining duplex stability and hybridization selectivity. TO fluorescence increased up to 26-fold upon hybridization. In contrast to most other probes; in which fluorescence is invariant once hybridization had occurred, the emission of TO-containing PNA probes is attenuated when forced to intercalate next to a mismatched base pair. The specificity of DNA detection is therefore not limited by the selectivity of probe-target binding and a DNA target can be distinguished from its single-base mutant under nonstringent hybridization conditions. This property should be of advantage for real-time quantitative PCR and nucleic acid detection within living cells.
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