Modification of oligonucleotides with dyes is a commonly used procedure in genomic assays, e.g., in the monitoring of real-time polymerase chain reaction. Most of the hybridization assays are based on the combination of a fluorophore with a quencher leading to the decrease of the fluorophore emission after detection of the target. The dark quenchers are a modern type of quenchers with no intrinsic fluorescence, which substantially simplifies the hybridization assays and increases their sensitivity.
During our studies of AzaPc, we systematically observed that alkylamino substituted derivatives even in the strictly monomeric form are lacking any fluorescent or photosensitizing properties typical for other AzaPc derivatives. Recently, we discovered and fully explained that ultrafast intramolecular charge transfer is responsible for the quenching of the S1 excited state of such AzaPc. This unique mechanism of S1 state deactivation, not yet observed at Pc derivatives, is responsible for the lack of fluorescence. These properties, together with strong absorption over a wide range of wavelengths, make alkylamino AzaPc ideal candidates for the modern dark quenchers. The experiments showed that AzaPc quenchers are highly efficient in combination with any fluorophore used in hybridization assays and for both quenching mechanisms (static and dynamic).
List of our important publications on the topic: