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 Nterminally 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.

Fluorogenic PNA probes

“…The use of pyrrolidinyl PNA probes gave an even better mismatch discrimination than conventional PNA probes [ 108 – 109 ]. The slow kinetics of the strand displacement involving highly stable PNA–DNA hybrids can be a major limitation of all the PNA-based strand displacement probes.…”

“…The equilibrium of the strand displacement reaction lies on the side of the more stable duplex, and so the original strand displacement probes are designed to have lower stabilities than the final duplexes. This can generally be achieved by employing a short complementary strand or by introducing mismatch pairs into the probe [ 109 ], but a C·I pseudocomplementary base pair, which is somewhat less stable than the C·G pair, has also been successfully employed in one case [ 111 ]. A PNA-based strand displacement probe was delivered into mammalian cells by employing cationic shell cross-linked nanoparticles, whereupon its application in the imaging of cellular mRNA expression was demonstrated [ 112 ].…”

Perspectives on conformationally constrained peptide nucleic acid (PNA): insights into the structural design, properties and applications

“…The fluorescence change was induced by the hybridization between the PNA probe and the DNA target that alters the environment of the dye. 134,276,277 Alternatively, a simple fluorophore-labeled PNA probe was used in combination with a quencher which can be present on the same PNA strand 166 or as a separate entity such as another quencher labeled PNA or DNA strand, 278 oligo(dG), 279 graphene oxide (GO), 280 and metal nanoparticles. 281 In all cases, the fluorescence of the free probe could be restored after hybridization with the correct DNA/RNA target which resulted in the separation of the probe and the quencher.…”

Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying fluorescence nucleobases

“…96 A strand displacement probe based on a fluorophore-labeled acpcPNA and a quencherlabeled DNA was also developed and demonstrated to show high mismatch discriminating ability. 97…”