Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No. 1, Yixueyuan Road, Yuzhong Dist, Chongqing, 400016, China.
Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No. 1, Yixueyuan Road, Yuzhong Dist, Chongqing, 400016, China.
Anal Chim Acta. 2022 Sep 22;1227:340266. doi: 10.1016/j.aca.2022.340266. Epub 2022 Aug 16.
Abnormal DNA methylation is closely related to the occurrence and development of many diseases. The determination of human DNA methyltransferase activity and the screening of its inhibitors are extreme important for the diagnosis and the treatment of methylation-related diseases in clinic. Most of the current detection methods have the disadvantages of sophisticated design, high cost and low detection limit. By combining T7 promoter-contained DNA probe as the substrate for methyltransferase with CRISPR/Cas13a sensing strategy, a novel fluorescent sensing platform is designed to achieve simple, specific, sensitive detection of bacteria DNA methyltransferase (DNA-(N-6-adenine)-methyltransferase, Dam MTase) and also human methyltransferase (DNA (cytosine-5)-methyltransferase 1, Dnmt1). A hairpin DNA probe designed for Dam MTase and a double strand DNA probe for Dnmt1 are both methylated followed by the methylation-dependent site-specific cleavage, which result a T7 promoter-contained product and a T7 promoter-free one to respectively open and close the transcription and subsequent CRISPR/Cas13a target-initiated cleavage of fluorescence-labeled reporter RNA. In virtue of the specificity of methylation-dependent cleavage of probe, the efficient transcription amplification and CRISPR/Cas13a sequence-specific sensing, this strategy exhibited remarkable specificity and sensitivity, with the limit of detection of 3.10 × 10 U/mL for Dam MTase. Moreover, Dnmt1 activity in MCF-7 cells was detected and the inhibition of Apt. #9 was evaluated. This strategy for methyltransferase detection is convenient and efficient for inhibitor discovery and early cancer diagnosis.
异常的 DNA 甲基化与许多疾病的发生和发展密切相关。测定人类 DNA 甲基转移酶的活性并筛选其抑制剂，对于临床中甲基化相关疾病的诊断和治疗至关重要。目前大多数检测方法存在设计复杂、成本高、检测限低等缺点。本研究将含 T7 启动子的 DNA 探针作为甲基转移酶的底物，与 CRISPR/Cas13a 传感策略相结合，设计了一种新型荧光传感平台，用于简单、特异、灵敏地检测细菌 DNA 甲基转移酶（DNA-(N-6-腺嘌呤)-甲基转移酶，Dam MTase）和人甲基转移酶（DNA (胞嘧啶-5)-甲基转移酶 1，Dnmt1）。设计了针对 Dam MTase 的发夹 DNA 探针和针对 Dnmt1 的双链 DNA 探针，均在甲基化后进行依赖于甲基化的位点特异性切割，分别产生含有 T7 启动子的产物和不含 T7 启动子的产物，从而开启和关闭转录以及随后的荧光标记报告 RNA 的 CRISPR/Cas13a 靶标起始切割。由于探针的依赖于甲基化的切割的特异性、有效的转录扩增和 CRISPR/Cas13a 序列特异性传感，该策略表现出显著的特异性和灵敏度，对 Dam MTase 的检测限为 3.10×10 U/mL。此外，还检测了 MCF-7 细胞中的 Dnmt1 活性，并评估了 Apt. #9 的抑制作用。该甲基转移酶检测策略方便、高效，可用于抑制剂发现和早期癌症诊断。
