Methoxy-Substituted Perylenediimide-Modified DNA for Enhanced Fluorescent Probes and Single-Nucleotide Discrimination

Artificial DNA molecules functionalized with fluorescent dyes are useful for developing fluorescent chemosensors and molecular imaging tools. Fluorescent nucleic acids have been developed based on the excellent fluorescent properties of perylenediimide (PDI); however, the electron transfer quenching of PDI by purine bases limits the sequence design of fluorescent probes. In this study, to compensate for the disadvantages of PDI due to electron transfer quenching, we used PDI with a methoxy group introduced into the perylene ring (PO), synthesized PO-substituted nucleic acids, and investigated their fluorescence properties. The results showed that PO in the DNA exhibited longer-wavelength fluorescence than unsubstituted PDI and displayed strong fluorescence when surrounded by AT base pairs. We also found that PO exhibits a fluorescence on-off response to the hybridization reaction and selective quenching by guanine bases in the vicinity of PO, which could be applied to fluorescent biosensors for single nucleotide identification and nucleic acid detection. Because of the high photostability of PO, single-molecule measurements were feasible, allowing confirmation of PO's fluorescence switching behavior of PO in response to DNA conformational changes and the presence of guanine bases at the single-molecule level using fluorescence correlation spectroscopy (FCS).