Highly Sensitive Fluorescence Detection of Global 5-Hydroxymethylcytosine from Nanogram Input with Strongly Emitting Copper Nanotags

作者：Zhenning Yu, Yanli Tong, Yuling Liang, Yun‐Da Li, Hongling Yang, Si‐Yang Liu, Yuzhi Xu, Zong Dai, Xiaoyong Zou · 发表于：Analytical Chemistry · 年份：2021 · DOI：10.1021/acs.analchem.1c03266 · 被引用次数：13 · 研究领域：Advanced biosensing and bioanalysis techniques、Epigenetics and DNA Methylation、HIV Research and Treatment

High Resolution Image Download MS PowerPoint Slide Quantitative analysis of 5-hydroxymethylcytosine (5hmC) has remarkable clinical significance to early cancer diagnosis; however, it is limited by the requirement in current assays for large amounts of starting material and expensive instruments requring expertise. Herein, we present a highly sensitive fluorescence method, termed hmC-TACN, for global 5hmC quantification from several nanogram inputs based on terminal deoxynucleotide transferase (TdT)-assisted formation of fluorescent copper (Cu) nanotags. In this method, 5hmC is labeled with click tags by T4 phage β-glucosyltransferase (β-GT) and cross-linked with a random DNA primer via click chemistry. TdT initiates the template-free extension along the primer at the modified 5hmC site and then generates a long polythymine (T) tail, which can template the production of strongly emitting Cu nanoparticles (CuNPs). Consequently, an intensely fluorescent tag containing numerous CuNPs can be labeled onto the 5hmC site, providing the sensitive quantification of 5hmC with a limit of detection (LOD) as low as 0.021% of total nucleotides ( S / N = 3). With only a 5 ng input (∼1000 cells) of genomic DNA, global 5hmC levels were accurately determined in mouse tissues, human cell lines (including normal and cancer cells of breast, lung, and liver), and urines of a bladder cancer patient and healthy control. Moreover, as few as 100 cells can also be distinguished between normal and cancer...