DNAzyme based three-way junction assay for antibody-free detection of locus-specific N6-methyladenosine modifications.
DNAzyme based three-way junction assay for antibody-free detection of locus-specific N6-methyladenosine modifications.
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DOI：
10.1016/j.bios.2021.113625
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发表时间：
2021-09
影响因子：
12.6
通讯作者：
Hongyan Yu;Qinli Pu;Zhi Weng;Xi Zhou;Junjie Li;Yujun Yang;Wang Luo;Yongcan Guo;Huajian Chen-Hua
中科院分区：
文献类型：
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作者：
Hongyan Yu;Qinli Pu;Zhi Weng;Xi Zhou;Junjie Li;Yujun Yang;Wang Luo;Yongcan Guo;Huajian Chen-Hua
N6-甲基腺苷（m6 A）是RNA中最丰富的转录后修饰，在生理过程和肿瘤发生中具有重要意义。然而，基因座特异性m6 A修饰水平的敏感和特异性定量仍然是一项具有挑战性的任务。在本工作中，一种新的m6 A敏感的DNAzyme被用来直接检测m6 A通过耦合与三向连接介导的等温指数CRISPR扩增反应的第一次。该方法建立在DNA酶的结合臂结合到特定位点及其核心结构催化未修饰的腺嘌呤而不是甲基化腺嘌呤的选择性切割的事实上。随后，通过三向连接的邻近效应来鉴定完整的RNA。通过SDA和EXPAR的组合用于信号放大，产生了大量的单链DNA产物。通过检测CRISPR Cas 12 a触发的荧光强度，记录产物的特异性实时曲线。结果，成功鉴定了丰度低至1%的甲基化靶标。此外，该策略可用于细胞RNA提取物的分析。结合用于定量检测RNA甲基化的电化学传感器，我们证明了所提出的策略的通用性。本研究为RNA中m6 A的分析提供了一个新的平台，并将促进其在临床疾病中的应用。
N6-methyladenosine (m6A) is the most abundant post-transcriptional modification in RNA and has important implications in physiological processes and tumor development. However, sensitive and specific quantification of locus-specific m6A modification levels remains a challenging task. In the present work, a novel m6A-sensitive DNAzyme was utilized to directly detect m6A by coupling with a three-way junction-mediated isothermal exponential CRISPR amplification reaction for the first time. This method was built on the fact that the binding arm of the DNAzyme bound to the specific site and its core structure catalyzed the selective cleavage of unmodified adenine instead of methylated adenines. Subsequently, the intact RNA was identified by the proximity effect of the three-way junction. Enormous amounts of single-stranded DNA products were generated through a combination of SDA and EXPAR for signal amplification. The specific real-time curve of products was recorded through detecting the fluorescence intensity triggered by CRISPR Cas12a. As a result, methylation target of abundance down to 1% was successfully identified. In addition, this strategy could be used for the analysis of cell RNA extracts. Combined with an electrochemical sensor for quantitative detection of RNA methylation, we demonstrated the generality of as-proposed strategy. We envision the present method would provide a new platform for the analysis of m6A in RNA and promote its application in clinical diseases.