Development of fluorescence pyrrolidinyl peptide nucleic acid as probes for determination of DNA sequences

Abstract

In this research, fluorescence peptide nucleic acid (PNA) probes that can change the fluorescence in response to the presence of correct DNA target were designed and evaluated. The PNA employed in this work is the pyrrolidinyl peptide nucleic acid with an alternating proline/2-aminocyclopentanecarboxylic acid backbone (acpcPNA) which shows superior binding to DNA in terms of stability and specificity compared to DNA and commercial PNA. In the first part, 5-(pyren-1-yl)uracil (UPy) PNA monomer was synthesized and incorporated into the acpcPNA. The UPy base in acpcPNA retained the ability to specifically recognize the base A in its complementary DNA strand. Furthermore, the fluorescence of the UPy base is significantly enhanced when the acpcPNA is hybridized to its complementary DNA target, irrespective of the nature of the flanking bases. The fluorescence enhancement is specific to the pairing between UPy and dA. In the second part, pyrene was covalently attached to N-terminal positions of acpcPNA or to internal positions of APC-modified acpcPNA through a flexible butyryl linker via acylation of the N-termini of acpcPNA or the pyrrolidine nitrogen atom of 3-aminopyrrolidine-4-carboxylic acid (APC)-modified acpcPNA. Hybridization of the terminally pyrene-modified PNA to its complementary DNA gave variable fluorescence change, depending on the original fluorescence of the single stranded PNA and the identity of the DNA base in vicinity of the pyrene label. Hybridization of the internally pyrene-labeled acpcPNA to its complementary DNA target resulted in a consistent large increase in fluorescence signal relative to the single stranded DNA. In the third part, the combination of terminally fluorophore-modified acpcPNAs (Flu- or TMR-acpcPNA) with a short quencher DNA were evaluated as strand-displacement probes for DNA sequence determination. In the presence of complementary DNA, the fluorescence was increased, but never returned to the original level due to incomplete strand displacement and also quenching by the DNA target. In all cases, the high specificity of the acpcPNA system allows unambiguous discrimination between the complementary and single mismatched DNA target.