Silver-Coordinated Watson–Crick Pairing-Driven Three-Dimensional DNA Walker for Locus-Specific Detection of Genomic N6-Methyladenine and N4-Methylcytosine at the Single-Molecule Level,Analytical Chemistry

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Silver-Coordinated Watson–Crick Pairing-Driven Three-Dimensional DNA Walker for Locus-Specific Detection of Genomic N6-Methyladenine and N4-Methylcytosine at the Single-Molecule Level
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-01-28 , DOI: 10.1021/acs.analchem.3c05184
Li-Juan Wang ₁ , Qian Liu ₁ , Ying-Ying Lu ₂ , Le Liang ₂ , Chun-Yang Zhang ₁

Affiliation

N⁶-Methyladenine (6mdA) and N⁴-methylcytosine (4mdC) are the two most dominant DNA modifications in both prokaryotes and eukaryotes, but standard hybridization-based techniques cannot be applied for the 6mdA/4mdC assay. Herein, we demonstrate the silver-coordinated Watson–Crick pairing-driven three-dimensional (3D) DNA walker for locus-specific detection of genomic 6mdA/4mdC at the single-molecule level. 6mdA-DNA and 4mdC-DNA can selectively hybridize with the binding probes (BP1 and BP2) to form 6mdA-DNA-BP1 and 4mdC-DNA-BP2 duplexes. The 6mdA-C/4mdC-A mismatches cannot be stabilized by Ag^I, and thus, 18-nt BP1/BP2 cannot be extended by the catalysis of KF exonuclease. Through toehold-mediated strand displacement (TMSD), the signal probe (SP1/SP2) functionalized on the gold nanoparticles (AuNPs) can competitively bind to BP1/BP2 in 6mdA-DNA-BP1/4mdC-DNA-BP2 duplex to obtain SP1–18-nt BP1 and SP2–18-nt BP2 duplexes. The resulting DNA duplexes can act as the substrates of lambda exonuclease, leading to the cleavage of SP1/SP2 and the release of Cy3/Cy5 and 18-nt BP1/BP2. The released 18-nt BP1/BP2 can subsequently serve as the walker DNA, moving along the surface of the AuNP to activate dynamic 3D DNA walking and releasing abundant Cy3/Cy5. The released Cy3/Cy5 can be quantified by single-molecule imaging. This nanosensor exhibits high sensitivity with a limit of detection (LOD) of 9.80 × 10^–15 M for 6mdA-DNA and 9.97 × 10^–15 M for 4mdC-DNA. It can discriminate 6mdA-/4mdC-DNA from unmodified genomic DNAs, distinguish 0.01% 6mdA-/4mdC-DNA from excess unmethylated DNAs, and quantify 6mdA-/4mdC-DNA at specific sites in genomic DNAs of liver cancer cells and Escherichia coli plasmid cloning vector, providing a new platform for locus-specific analysis of 6mdA/4mdC in genomic DNAs.

中文翻译：

银配位沃森-克里克配对驱动的三维 DNA Walker 用于单分子水平基因组 N6-甲基腺嘌呤和 N4-甲基胞嘧啶的位点特异性检测

N ^{6 -}甲基腺嘌呤 (6mdA) 和N ^{4 -甲基}胞嘧啶 (4mdC) 是原核生物和真核生物中两种最主要的 DNA 修饰，但基于标准杂交的技术不能应用于 6mdA/4mdC 测定。在此，我们展示了银配位的 Watson-Crick 配对驱动的三维 (3D) DNA 步行器，用于在单分子水平上对基因组 6mdA/4mdC 进行位点特异性检测。 6mdA-DNA和4mdC-DNA可以选择性地与结合探针（BP1和BP2）杂交，形成6mdA-DNA-BP1和4mdC-DNA-BP2双链体。 Ag ^I无法稳定 6mdA-C/4mdC-A 错配，因此 KF 核酸外切酶无法催化 18-nt BP1/BP2 延伸。通过立足点介导的链置换（TMSD），在金纳米颗粒（AuNPs）上功能化的信号探针（SP1/SP2）可以竞争性地结合6mdA-DNA-BP1/4mdC-DNA-BP2双链体中的BP1/BP2，从而获得SP1– 18-nt BP1 和 SP2–18-nt BP2 双链体。产生的 DNA 双链体可以作为 lambda 核酸外切酶的底物，导致 SP1/SP2 裂解并释放 Cy3/Cy5 和 18-nt BP1/BP2。释放的 18-nt BP1/BP2 随后可以作为步行 DNA，沿着 AuNP 表面移动，激活动态 3D DNA 步行并释放丰富的 Cy3/Cy5。释放的 Cy3/Cy5 可以通过单分子成像进行定量。该纳米传感器具有高灵敏度，6mdA-DNA 的检测限 (LOD) 为 9.80 × 10 ^–15 M，4mdC-DNA 的检测限为 9.97 × 10 ^–15 M。它可以区分 6mdA-/4mdC-DNA 和未修饰的基因组 DNA，区分 0。从过量的未甲基化DNA中提取01% 6mdA-/4mdC-DNA，并定量肝癌细胞和大肠杆菌质粒克隆载体基因组DNA中特定位点的6mdA-/4mdC-DNA，为6mdA/位点特异性分析提供新平台基因组 DNA 中的 4mdC。

更新日期：2024-01-28

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