Probing the kinetics of SYTOX orange stain binding to double-stranded DNA with implications for DNA analysis

Rapid binding kinetics of SYTOX Orange stain with double-stranded DNA (dsDNA) was revealed on the DNA fragment sizing flow cytometer. We demonstrated for the first time that the dye molecules could be adsorbed onto the capillary surface and native DNA fragments can be dynamically stained while passing through the capillary. High-quality burst size distribution histograms were obtained for DNA samples analyzed immediately after staining, dilution, or mixing. These observations indicated that rapid interactions exist between SYTOX Orange dye molecules and dsDNA. A stopped-flow fluorescence apparatus was set up to capture the fast association traces of intercalating dyes binding to dsDNA. Kinetic equations were derived to fit the association curves for determination of association rates and to model the dynamic staining, dilution, and mixing processes of DNA samples stained with intercalating dyes. The measured association rates for both SYTOX Orange and PicoGreen stains intercalating into dsDNA were on the order of 10(8) M-1 s(-1), suggesting a diffusion-controlled process. Simulations indicate that reequilibration can be reached in seconds upon staining, dilution, or mixing. Insight into the kinetics of DNA binding dyes will help implement efficient sample-handling practices in DNA analysis, including DNA fragment sizing flow cytometry.