Effects of Mg2+ and ATP on YOYO-1 labeling of genomic DNA in single molecule experiments
Journal article, 2025

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.

Nanofluidic channels

YOYO-1

Single molecule analysis

Mg2+

DNA

ATP

Author

Carl Ivar Möller

Chalmers, Life Sciences, Chemical Biology

Dennis Raphael Winter

Chalmers, Life Sciences, Chemical Biology

Radhika Nambannor Kunnath

Chalmers, Life Sciences, Chemical Biology

Sriram Kesarimangalam

Chalmers, Life Sciences, Chemical Biology

Fredrik Westerlund

Chalmers, Life Sciences, Chemical Biology

Biochemistry and Biophysics Reports

2405-5808 (eISSN)

Vol. 44 102248

Subject Categories (SSIF 2025)

Molecular Biology

Condensed Matter Physics

DOI

10.1016/j.bbrep.2025.102248

More information

Latest update

9/19/2025