Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University , Jinan 250014, China.
Nantou High School Shenzhen , Shenzhen, 518052, China.
Anal Chem. 2017 Nov 21;89(22):12408-12415. doi: 10.1021/acs.analchem.7b03490. Epub 2017 Nov 7.
DNA methyltransferases (MTases) may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-l-methionine to target cytosine/adenine. The aberrant DNA methylation is linked to the abnormal DNA MTase activity, and some DNA MTases have become promising targets of anticancer/antimicrobial drugs. However, the reported DNA MTase assays often involve laborious operation, expensive instruments, and radio-labeled substrates. Here, we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase (Dam) on the basis of terminal deoxynucleotidyl transferase (TdT)-activated Endonuclease IV (Endo IV)-assisted hyperbranched amplification. We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH-modified 3' end for the prevention of nonspecific amplification. The substrate may be methylated by Dam and subsequently cleaved by DpnI, producing three single-stranded DNAs, two of which with 3'-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal. Because high exactitude of TdT enables the amplification only in the presence of free 3'-OH termini and Endo IV only hydrolyzes the intact apurinic/apyrimidinic sites in double-stranded DNAs, zero background signal can be achieved. This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61 × 10 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in disease diagnosis and drug development.
DNA 甲基转移酶（MTases）可能特异性识别短的回文序列，并将 S-腺苷甲硫氨酸中的甲基转移到靶标胞嘧啶/腺嘌呤上。异常的 DNA 甲基化与异常的 DNA MTase 活性有关，一些 DNA MTases 已成为有前途的抗癌/抗菌药物靶点。然而，已报道的 DNA MTase 测定法通常涉及繁琐的操作、昂贵的仪器和放射性标记的底物。在这里，我们基于末端脱氧核苷酸转移酶（TdT）激活的内切核酸酶 IV（Endo IV）辅助超支化扩增，开发了一种简单且无标记的荧光法来灵敏地检测 DNA 腺嘌呤甲基转移酶（Dam）。我们设计了一种发夹探针，其茎部具有回文序列作为底物，并且 3'端经过 NH 修饰以防止非特异性扩增。该底物可被 Dam 甲基化，随后被 DpnI 切割，产生三条单链 DNA，其中两条具有 3'-OH 末端，可通过超支化扩增进行扩增，从而产生独特的荧光信号。由于 TdT 的高精度仅允许在存在游离 3'-OH 末端的情况下进行扩增，并且 Endo IV 仅水解双链 DNA 中的完整无嘌呤/无嘧啶位点，因此可以实现零背景信号。该方法具有出色的选择性和高灵敏度，对纯 Dam 的检测限为 0.003 U/mL，对大肠杆菌细胞中的 Dam 的检测限为 9.61×10^-3 mg/mL。此外，它可用于筛选 Dam 抑制剂，在疾病诊断和药物开发方面具有巨大潜力。
