Quenching of fluorescently labeled pyrrolidinyl peptide nucleic acid by oligodeoxyguanosine and its application in DNA sensing.

Abstract

Quenching by nucleobases can significantly affect the fluorescence properties of many fluorophores. The quenching efficiency depends on the electronic properties of the fluorophore and adjacent nucleobases. In this study, we present a hitherto unreported high-efficiency quenching (up to 90%) of various fluorescently labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probes by oligodeoxyguanosine (dGX). The quenching principle relies on the electrostatic interaction between the positively charged lysine-modified acpcPNA probe and the negatively charged oligodeoxyguanosine. The addition of stoichiometric quantities of a DNA target with the sequence complementary to the PNA probe restored the fluorescence to the original level. This was explained by the binding of the DNA to the PNA via a specific base pairing, which resulted in the separation of the oligodeoxyguanosine quencher from the fluorophore. Much less fluorescence restoration was observed in the DNA containing one or more mismatched bases. Applications of the oligodeoxyguanosine-quenched PNA probes for DNA sequence determination, including in multiplex formats, are demonstrated. The performance in terms of sensitivity and mismatch discrimination is comparable to classical PNA-based molecular beacons but without the need for double-labeling, which is expensive and presents solubility issues, or a dedicated quencher probe. This exemplifies a novel use of the unique electrostatic properties of PNA to develop a DNA sensing platform for DNA sequence determination.