Fluorescence detection of DNA with a single-base mismatch by a Tm-independent peptide nucleic acid (PNA) twin probe†

There is a need to develop efficient methods for detecting target nucleic acids to enable the rapid diagnosis and early treatment of diseases. We previously demonstrated that a peptide nucleic acid (PNA) twin probe, consisting of two PNAs each containing a fluorescent dye, with pyrene at one end, detects target DNA sequence-specifically through pyrene excimer emission. In this study, to advance the development of this probe system, we further investigated the fluorescence properties of the PNA twin probe and , and found that the excimer fluorescence was significantly reduced when a mismatched base in the DNA sequence was present at the site of closest to the pyrene. In other words, this probe was found to detect single-base mismatches without taking into account the thermal stability of the PNA/DNA hybrid. The detection limit of this PNA twin probe for the single-base-mismatched DNA was 2.7 nM. In the future, this probe should lead to a method to detect point mutations in endogenous nucleic acids within cells.