Dual sensing of hairpin and quadruplex DNA structures using multicolored peptide nucleic acid fluorescent probes

Synthesis of water-soluble 5-mer peptide nucleic acids (PNAs) functionalized at their 5'- and 3'-ends with two original precursors of pentamethine cyanine dye synthesis is reported. The successful use of these PNA probes for sensing DNA hairpin structures in vitro was also demonstrated where specific hairpin formation was associated with the appearance of a characteristic fluorescence signal at 660 nm. A comparative study between three different strategies where PNAs were targeting either the stem or the loop of the hairpin was carried out. Best sensitivity was obtained using PNA sequences complementary to the loop sequence and directing both functional moieties toward the base of loop. Unprecedented proof-of-concept for the simultaneous sensing of hairpin and quadruplex DNAs with a nonoverlapping two-color system (C3 and C5) is also demonstrated.