Pyrrolidinyl peptide nucleic acid terminally labeled with fluorophore and end-stacking quencher as a probe for highly specific DNA sequence discrimination

Abstract

Peptide nucleic acid (PNA) labeled with a dye/quencher pair is useful as a high-performance fluorescence probe for DNA sequence determination. Previous work has mainly focused on placing the dye/quencher at different ends of the PNA strand. In this work, we propose a new pyrrolidinyl PNA probe bearing a fluorescent dye and an anthraquinone quencher at the same end of the PNA molecule. The single-stranded PNA probe exhibits a weak fluorescence as a result of close contact between the dye and the quencher. End-stacking of the anthraquinone to the terminal base pair of the PNA·DNA duplex disrupts the fluorophore-quencher interaction, resulting in a large fluorescence increase in the presence of complementary DNA. The high specificity of the present PNA probe allows multicolor, multiplex detection of single mismatched DNA targets. In addition, direct detection of double stranded DNA target by double duplex invasion was demonstrated.