A simple fluorescent assay for the detection of peptide 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.

Exploring Nucleobase Modifications in Oligonucleotide Analogues for Use as Environmentally Responsive Fluorophores and Beyond

“…[117] The fluorescence turn-on in single stranded PhpC compared to double stranded PhpC was exploited to monitor double duplex invasion of DNA by pseudo complementary PNAs. [118] More recently, it was discovered that a PhpC PNA submonomer caused destabilization of G-quadruplex structures by facilitating the activity of a G4-helicase. [119] This is a particularly exciting observation because over representation of G-quadruplexes is related to genetic age-related disorders and very few Gquadruplex disruptors are known.…”

“…When incorporated into PNA oligomers, the intrinsic fluorescence of boPhpC has been used to study cellular localization of anti‐sense agents directed at Huntingtin mRNA [116] and anti‐microRNA‐122 [117] . The fluorescence turn‐on in single stranded PhpC compared to double stranded PhpC was exploited to monitor double duplex invasion of DNA by pseudo complementary PNAs [118] . More recently, it was discovered that a PhpC PNA submonomer caused destabilization of G‐quadruplex structures by facilitating the activity of a G4‐helicase [119] .…”