化学
底漆(化妆品)
DNA
寡核苷酸
清脆的
分子生物学
甲基转移酶
DNA聚合酶
聚合酶
生物化学
甲基化
生物
基因
有机化学
作者
Human Sun,Shiying Zhou,Yin Liu,Peng Lü,Na Qi,Guixue Wang,Mei Yang,Danqun Huo,Changjun Hou
标识
DOI:10.1016/j.aca.2022.340732
摘要
DNA methyltransferase (DNA MTase) catalyzes the process of DNA methylation, and the aberrant DNA MTase activity is closely associated with cancer incidence and progression. Inspired by the exponential amplification reaction (EXPAR) characteristics, we developed an EXPAR-initiated CRISPR/Cas12a (EIC) strategy for sensitively detecting DNA MTase activity. A hairpin probe (HP) was designed with a palindromic sequence in the stem as substrate and NH2-modified 3' end to prevent nonspecific amplification. HP could be methylated by DNA adenine methyltransferase (Dam MTase) and then digested by DpnI to generate an oligonucleotide that can serve as an EXPAR primer. With the assistance of Nt.BstNBI nicking enzyme and Vent(exo-) polymerase, this primer bound to template and induced EXPAR. Interestingly, the product of Cycle 1 in EXPAR can function as primer to initiate Cycle 2. Both EXPAR products can further activate the collateral cleavage of CRISPR/Cas12a-crRNA, resulting in the fragmentation of fluorescence reporters and fluorescence recovery. Due to the highly efficient amplification (about 5 times signal-to-noise of SDA) and the robust trans-cleavage of CRISPR/Cas12a, the EIC system owned an extreme limit of detection (LOD) of 2 × 10-4 U/mL and a broad detection range from 2 × 10-4 to 10 U/mL for Dam MTase. In addition, this method has succeeded in inhibitor screening and evaluation, showing magnificent promise in drug discovery and cancer therapy.
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