Preparation and Performance Evaluation of a Pyrrolidinyl Peptide Nucleic‐Acid‐Based Displacement Probe as a DNA Sensor

Abstract A new displacement probe based on pyrrolidinyl peptide nucleic acid was designed and evaluated for DNA sequence recognition. The probe was prepared by combining an N‐terminally fluorophore‐modified pyrrolidinyl peptide nucleic acid (Flu‐ or TMR‐acpcPNA) and a 3’‐Dabcyl‐modified DNA as a quencher. Fluorescence studies showed that the fluorophore in the acpcPNA strand was efficiently quenched by the quencher strand. After some optimisation, the fluorescence was significantly restored upon the addition of the complementary DNA target, while the fluorescence stayed at a low level with the addition of mismatched DNA. Even with double‐stranded DNA analytes, the high specificity of the PNA‐based displacement probes allowed unambiguous discrimination between complementary and single mismatched DNA targets. Furthermore, immobilisation of the probes onto agarose resin could also recognise only the complementary DNA, thereby demonstrating its potential as a practical DNA sensor.