Effects of Mg²⁺ 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 Mg²⁺ as a cofactor. Mg²⁺ 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 Mg²⁺ ions and ATP on YOYO-1 labeled genomic DNA and shows, via single molecule experiments in nanochannels, that Mg²⁺ 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 Mg²⁺, but only at comparable concentrations. Additionally, while increasing the photobleaching rate, Mg²⁺ 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 Mg²⁺ causes dissociation of YOYO-1 from DNA. These findings demonstrate that the addition of Mg²⁺ and ATP poses challenges in DNA-protein studies using nanofluidics, which can be mitigated by optimizing experimental conditions.