Identification and Quantification of Locus-Specific 8-Oxo-7,8-dihydroguanine in DNA at Ultrahigh Resolution Based on G-Triplex-Assisted Rolling Circle Amplification,Analytical Chemistry

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Identification and Quantification of Locus-Specific 8-Oxo-7,8-dihydroguanine in DNA at Ultrahigh Resolution Based on G-Triplex-Assisted Rolling Circle Amplification
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-12-27 , DOI: 10.1021/acs.analchem.3c04498
Jia-Hui Dong ₁ , Run-Hong Zhang ₁ , Ling-Li Zhao ₂ , Chen-Yu Xue ₃ , Hui-Yu Pan ₁ , Xin-Ying Zhong ₄ , Ying-Lin Zhou ₁ , Xin-Xiang Zhang ₁

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Damage of reactive oxygen species to various molecules such as DNA has been related to many chronic and degenerative human diseases, aging, and even cancer. 8-Oxo-7,8-dihydroguanine (OG), the most significant oxidation product of guanine (G), has become a biomarker of oxidative stress as well as gene regulation. The positive effect of OG in activating transcription and the negative effect in inducing mutation are a double-edged sword; thus, site-specific quantification is helpful to quickly reveal the functional mechanism of OG at hotspots. Due to the possible biological effects of OG at extremely low abundance in the genome, the monitoring of OG is vulnerable to signal interference from a large amount of G. Herein, based on rolling circle amplification-induced G-triplex formation and Thioflavin T fluorescence enhancement, an ultrasensitive strategy for locus-specific OG quantification was constructed. Owing to the difference in the hydrogen-bonding pattern between OG and G, the nonspecific background signal of G sites was completely suppressed through enzymatic ligation of DNA probes and the triggered specificity of rolling circle amplification. After the signal amplification strategy was optimized, the high detection sensitivity of OG sites with an ultralow detection limit of 0.18 amol was achieved. Under the interference of G sites, as little as 0.05% of OG-containing DNA was first distinguished. This method was further used for qualitative and quantitative monitoring of locus-specific OG in genomic DNA under oxidative stress and identification of key OG sites with biological function.

中文翻译：

基于 G-Triplex 辅助滚环扩增超高分辨率鉴定和定量 DNA 中位点特异性 8-Oxo-7,8-二氢鸟嘌呤

活性氧对DNA等多种分子的损伤与许多慢性和退行性人类疾病、衰老甚至癌症有关。 8-Oxo-7,8-二氢鸟嘌呤 (OG) 是鸟嘌呤 (G) 最重要的氧化产物，已成为氧化应激和基因调控的生物标志物。 OG激活转录的积极作用和诱导突变的消极作用是一把双刃剑；因此，位点特异性定量有助于快速揭示OG在热点处的功能机制。由于OG在基因组中丰度极低时可能产生生物效应，OG的监测容易受到大量G的信号干扰。在此，基于滚环扩增诱导的G-三链体形成和硫磺素T荧光增强，构建了一种用于位点特异性 OG 定量的超灵敏策略。由于OG和G之间氢键模式的差异，通过DNA探针的酶连接和滚环扩增的触发特异性，G位点的非特异性背景信号被完全抑制。优化信号放大策略后，实现了OG位点的高检测灵敏度，超低检出限为0.18 amol。在G位点的干扰下，只有0.05%的含OG DNA首次被识别出来。该方法进一步用于氧化应激下基因组DNA中位点特异性OG的定性和定量监测以及具有生物学功能的关键OG位点的鉴定。

更新日期：2023-12-27

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