化学
末端脱氧核苷酸转移酶
DNA
甲基转移酶
胞嘧啶
分子生物学
回文序列
分子信标
核酸内切酶
底漆(化妆品)
DNA聚合酶
检出限
生物物理学
寡核苷酸
生物化学
甲基化
生物
标记法
回文
清脆的
基因
色谱法
有机化学
细胞凋亡
作者
Yan Zhang,Xinyan Wang,Qianyi Zhang,Chunyang Zhang
出处
期刊:Analytical Chemistry
[American Chemical Society]
日期:2017-11-07
卷期号:89 (22): 12408-12415
被引量:45
标识
DOI:10.1021/acs.analchem.7b03490
摘要
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 NH2-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 × 10–6 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.
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