DNA methylation is an important epigenetic modification in human genomes. Herein, we develop a single quantum dot (QD)-based nanosensor for sensitive detection of DNA methylation at both CpG and non-CpG sites using tricyclic ligation chain reaction (LCR)-mediated QD-based fluorescence resonance energy transfer (FRET). We design two sets of DNA probes (X and Y, X′ and Y′) for methylated DNA assay. In the presence of thermostable DNA ligase, probes X and Y may adjacently hybridize with the methylated DNA to obtain the ligated XY products which may function as the templates for probes X′ and Y′ to generate the X′Y′ products. The resultant X′Y′ products may in turn act as the templates to ligate probes X and Y for the generation of XY products, consequently inducing tricyclic LCR amplification under thermal denaturation conditions to generate a large number of XY products. The subsequent hybridization of XY products with the capture and reporter probes results in the formation of sandwich hybrids which may assemble on the 605QD surface to obtain 605QD–oligonucleotide–Cy5 nanostructures, inducing efficient FRET from the 605QD to Cy5 and the emission of Cy5. This nanosensor can detect DNA methylation at single 5-methylcytosine (5-mC) resolution with a detection limit of as low as 1.0 aM and a large dynamic range of 7 orders of magnitude. Moreover, this nanosensor can distinguish as low as a 0.01% methylation level, and it can detect DNA methylation in human lung cancer cells as well, holding great potential for accurate epigenetic evaluation and early cancer diagnosis.

中文翻译：

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基于单量子点的纳米传感器，可灵敏检测 CpG 和非 CpG 位点的 5-甲基胞嘧啶†

DNA甲基化是人类基因组中重要的表观遗传修饰。在此，我们开发了一种基于单量子点 (QD) 的纳米传感器，用于使用三环连接链反应 (LCR) 介导的基于 QD 的荧光共振能量转移 (FRET) 灵敏检测 CpG 和非 CpG 位点的 DNA 甲基化。我们设计了两组 DNA 探针（X 和 Y，X' 和 Y'）用于甲基化 DNA 测定。在热稳定性DNA连接酶存在下，探针X和Y可以与甲基化DNA相邻杂交以获得连接的XY产物，该产物可以作为探针X'和Y'的模板，产生X'Y'产物。所得的X'Y'产物反过来可以作为模板连接探针X和Y以产生XY产物，从而在热变性条件下诱导三环LCR扩增以产生大量XY产物。随后 XY 产物与捕获探针和报告探针的杂交导致夹心杂交体的形成，该夹心杂交体可在 605QD 表面组装以获得 605QD-寡核苷酸-Cy5 纳米结构，从而诱导从 605QD 到 Cy5 的有效 FRET 以及 Cy5 的发射。该纳米传感器能够以单 5-甲基胞嘧啶 (5-mC) 分辨率检测 DNA 甲基化，检测限低至 1.0 aM，动态范围高达 7 个数量级。此外，这种纳米传感器可以区分低至0.01%的甲基化水平，并且还可以检测人类肺癌细胞中的DNA甲基化，在准确的表观遗传学评估和早期癌症诊断方面具有巨大的潜力。