Dual-pyrene-labeled pyrrolidinyl peptide nucleic acid probes

Abstract

The main objective of this research is to design a new excimer-monomer switching fluorescence PNA probe for DNA sequence detection. Pyrrolidinyl peptide nucleic acid bearing an α,β-dipeptide backbone (acpcPNA) deriving from D-prolyl-2-aminocyclopetane carboxylic acid was synthesized and site-specifically labeled at the backbone with two pyrene units. In single stranded form, the dual pyrene-labeled acpcPNA probe folded itself to bury the pyrene labels from the hydrophilic aqueous environment, resulting in the excimer emission at 470 nm. After hybridization with target DNA, the PNADNA duplex formed and the pyrene labels were forced away from each other, resulting in the monomer emission at 380 nm. To evaluate the efficiency of dual pyrene-labeled acpcPNA probe, the switching ratio was introduced. This was defined as a ratio of the monomer to excimer emissions in the double strand [F380/F470(ds)] divided by the single strand [F380/F470(ss)] forms. The higher the switching ration, the more efficient is the probe. The switching ratios of ~5-30 were observed for all acpcPNA probes. The best ratio of ~30 was obtained in a homothymine sequence with the pyrenebutyl labels located 5-base apart of pyrenes. Lower switching ratios ~5–8 were observed with mix-base DNA sequences. Excellent discrimination between complementary and single mismatched DNA targets was achieved for all probes. The results showed that these dual pyrene-labeled acpcPNA probes are potentially useful for DNA sequence determination. An application of the probe in monitoring the DNA duplex invasion by acpcPNA was also demonstrated.