Switchable inhibitory behavior of divalent magnesium ion in DNA hybridization-based gene quantification
Switchable inhibitory behavior of divalent magnesium ion in DNA hybridization-based gene quantification
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DOI：
10.1039/d2an01164f
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发表时间：
2022-09-18
期刊：
影响因子：
4.2
通讯作者：
Son，Ahjeong
中科院分区：
文献类型：
--
作者：
Jin，Hyowon;Lim，Hyun Jeong;Son，Ahjeong
与二价阳离子只会导致基于DNA杂交的分析低估基因定量的理解相反，我们发现镁离子在不同浓度下可能导致低估或高估。其可切换的抑制行为可能是由于其刚性的第一溶剂化(水合)壳，因此它倾向于与DNA形成非直接结合。在低浓度时，它通过占据杂交位点而导致低估。在高浓度时，它会导致探针、信号和靶DNA通过库仑力非特异性地聚集。通过使用基因定量分析(纳米基因分析)在一定的镁浓度范围内定量目标DNA，观察到目标单链DNA和双链DNA的镁离子拐点浓度均为∼10−3 M。利用场发射扫描电子显微镜(FE-SEM)、能谱仪(EDS)和傅立叶变换红外光谱(FT-IR)观察了模拟靶DNA存在的配合物中镁离子诱导的非特异性结合。与另外两个二价阳离子一起，通过Zeta电位测量和纳米基因分析对它们进行了进一步的检测。这项研究揭示了镁离子在实现准确的基因定量方面的重要性。通过更好地从机理上理解这一现象，将有可能开发出减轻镁离子对基于DNA杂交的基因定量的影响的策略。
Contrary to the understanding that divalent cations only result in under-estimation of gene quantification via DNA hybridization-based assays, we have discovered that Mg2+ could cause either under or over-estimation at different concentrations. Its switchable inhibitory behavior is likely due to its rigid first solvation (hydrated) shell and hence it is inclined to form non-direct binding with DNA. At low concentrations, it caused under-estimation by occupying the hybridization sites. At high concentrations, it caused probe, signaling and target DNA to aggregate non-specifically via Coulomb forces. By quantifying target DNAs at a range of Mg2+ concentrations using a gene quantification assay (NanoGene assay), a Mg2+ inflection concentration of ∼10−3 M was observed for both target ssDNA and dsDNA. Field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectroscopy (FT-IR) were employed to observe Mg2+-induced non-specific binding in the complexes that mimicked the presence of target DNA. Together with two other divalent cations Ca2+ and Cu2+, they were further examined via zeta potential measurements as well as NanoGene assay. This study revealed the importance of Mg2+ in achieving accurate gene quantification. Through a better mechanistic understanding of this phenomenon, it will be possible to develop strategies to mitigate the impact of Mg2+ on DNA hybridization-based gene quantification.