Effects of Mg and ATP on YOYO-1 labeling of genomic DNA in single molecule experiments.

Nanofluidic channels have emerged as a suitable tool to study DNA-protein interactions. Many DNA-interacting proteins require ATP to fully function and use Mg as a cofactor. Mg and ATP are however also known to influence the binding of dyes, such as the commonly used YOYO-1, to DNA. This study investigates the effects of Mg ions and ATP on YOYO-1 labeled genomic DNA and shows, via single molecule experiments in nanochannels, that Mg reduces the fluorescence intensity of YOYO-1 labeled DNA, as well as the extension of the DNA, at both low and high dye loadings. When combined, ATP counteracts the loss of fluorescence caused by Mg, but only at comparable concentrations. Additionally, while increasing the photobleaching rate, Mg delays dye-mediated photolytic DNA damage, reducing DNA fragmentation in the nanofluidic channels. Determination of the apparent binding constant by bulk measurements corroborates the single molecule observations, suggesting that Mg causes dissociation of YOYO-1 from DNA. These findings demonstrate that the addition of Mg and ATP poses challenges in DNA-protein studies using nanofluidics, which can be mitigated by optimizing experimental conditions.