Möller Carl, Winter Dennis, Nambannor Kunnath Radhika, Kk Sriram, Westerlund Fredrik
Department of Life Sciences, Chalmers University of Technology, Gothenburg, SE, 412 96, Sweden.
Biochem Biophys Rep. 2025 Sep 10;44:102248. doi: 10.1016/j.bbrep.2025.102248. eCollection 2025 Dec.
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.
纳米流体通道已成为研究DNA-蛋白质相互作用的合适工具。许多与DNA相互作用的蛋白质需要ATP才能充分发挥功能，并将镁作为辅助因子。然而，已知镁和ATP也会影响染料（如常用的YOYO-1）与DNA的结合。本研究调查了镁离子和ATP对YOYO-1标记的基因组DNA的影响，并通过纳米通道中的单分子实验表明，在低染料负载和高染料负载情况下，镁都会降低YOYO-1标记的DNA的荧光强度以及DNA的伸展。当ATP与镁结合时，它会抵消镁引起的荧光损失，但仅在浓度相当的情况下。此外，虽然镁会提高光漂白速率，但它会延迟染料介导的光解DNA损伤，减少纳米流体通道中的DNA片段化。通过大量测量确定的表观结合常数证实了单分子观察结果，表明镁会导致YOYO-1从DNA上解离。这些发现表明，在使用纳米流体技术进行DNA-蛋白质研究时，添加镁和ATP会带来挑战，通过优化实验条件可以减轻这些挑战。
