Forced Intercalation Probes (FIT Probes): Thiazole Orange as a Fluorescent Base in Peptide Nucleic Acids for Homogeneous Single‐Nucleotide‐Polymorphism Detection

Abstract 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.