SYBR Green I: Fluorescence Properties and Interaction with DNA

In this study, we have investigated the fluores- cence properties of SYBR Green I (SG) dye and its interac- tion with double-stranded DNA (dsDNA). SG/dsDNA complexes were studied using various spectroscopic techni- ques, including fluorescence resonance energy transfer and time-resolved fluorescence techniques. It is shown that SG quenching in the free state has an intrinsic intramolecular origin; thus, the observed >1,000-fold SG fluorescence en- hancement in complex with DNA can be explained by a dampening of its intra-molecular motions. Analysis of the obtained SG/DNA binding isotherms in solutions of differ- ent ionic strength and of SG/DNA association in the pres- ence of a DNA minor groove binder, Hoechst 33258, revealed multiple modes of interaction of SG inner groups with DNA. In addition to interaction within the DNA minor groove, both intercalation between base pairs and stabi- lization of the electrostatic SG/DNA complex contribut- ed to increased SG affinity to double-stranded DNA. We show that both fluorescence and the excited state lifetime of SG dramatically increase in viscous solvents, demonstrating an approximate 200-fold enhancement in 100 % glycerol, compared to water, which also makes SG a prospective fluorescent viscosity probe. A pro- posed structural model of the SG/DNA complex is compared and discussed with results recently reported for the closely related PicoGreen chromophore.