清脆的
DNA甲基化
反式激活crRNA
DNA
费斯特共振能量转移
甲基化
计算生物学
多路复用
核酸
Cas9
寡核苷酸
化学
生物
组合化学
分子生物学
遗传学
荧光
生物化学
基因
基因表达
物理
量子力学
作者
Lihua Ding,Shengnan Cao,Chenling Qu,Yongjun Wu,Songcheng Yu
出处
期刊:ACS Sensors
[American Chemical Society]
日期:2024-04-04
卷期号:9 (4): 1877-1885
被引量:2
标识
DOI:10.1021/acssensors.3c02571
摘要
The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Förster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies.
科研通智能强力驱动
Strongly Powered by AbleSci AI