Label-Free in Situ Monitoring of the DNA Hybridization Chain Reaction by Using Sequence-Selective Minor-Groove-Binding Fluorophores

A new label-free in situ monitoring system for the hybridization chain reaction (HCR) based on DNA minor-groove-binding fluorophores [Hoechst 33258 (Hoe) or quinone cyanine-dithiazole (QCy-DT)] has been developed. Use of two unmodified hairpin oligodeoxyribonucleotides containing incomplete double-stranded AATT sequences enabled target-dependent formation of probe binding sitesthat is, AATT double strandin the HCR product, together with fluorescence enhancement of minor-groove-binding fluorophores in situ. This system allows target DNA to be detected through the fluorescence enhancement of Hoe and QCy-DT in real time and in situ. Further development of a label-free, isothermal detection system might provide a cost-effective and user-friendly method for nucleic acid detection.