A simple fluorescent assay for the detection of p eptide nucleic acid ‐directed double strand duplex invasion

Abstract Peptide nucleic acid (PNA) is a mimic of nucleic acids that is able to bind complementary oligonucleotides with high affinity and excellent selectivity. As such, the use of PNA has been proposed in numerous applications in biochemistry, medicine, and biotechnology. Sequences of pseudo‐complementary PNAs containing diaminopurine (D)‐2‐thiouracil (SU) base pairs bind to complementary regions within double‐stranded DNA targets. This type of binding is termed “double duplex invasion” and involves unwinding of the duplex accompanied by simultaneous hybridization of both DNA strands by the two pseudo‐complementary PNAs. In this study, a simple method of assaying DNA strand invasion by pseudo‐complementary PNAs was developed. This method is based on the incorporation of a single fluorescent cytidine analog, phenylpyrrolocytidine (PhpC), into the double‐stranded DNA target such that upon invasion by PNA, the PhpC is displaced to a single‐stranded region resulting in the turn‐on of fluorescence emission. This fluorescent assay is applicable to studies both at equilibrium and approach‐to‐equilibrium (time‐dependent) conditions.