Construction Of An Enzyme-Free Initiator-Replicated Hybridization Chain Reaction Circuit For Amplified Methyltransferase Evaluation And Inhibitor Assay

ANALYTICAL CHEMISTRY(2021)

引用 31|浏览43
暂无评分
摘要
The enzyme-free nucleic acid amplification circuit, for example, hybridization chain reaction (HCR), has paved a broad avenue for evaluating various enzyme-involved biotransformations, including DNA methyltransferases (MTases). The nonenzymatic MTase-sensing platform has supplemented a versatile toolbox for monitoring aberrant methylation in intricate biological samples, yet their amplification efficiency is always constrained by the initiator-depletion paradigm. Herein, the autonomously initiator-replicated HCR (IR-HCR) was developed as a versatile amplification system for detecting MTase with similar to 100-fold sensitivity of the conventional HCR system. The initiator I-triggered HCR leads the assembly of a tandem DNAzyme concatemer that cleaves its substrate. This leads to the cyclic replication of a new initiator I for reversely motivating the initial HCR circuit, resulting in a dramatic Forster resonance energy transfer (FRET) readout. Without M.SssI MTase, hairpin H-M can be recognized and digested by restriction endonuclease HpaII to release initiator I for stimulating a high FRET signal. While the M.SssI-methylated H-M prohibits the HpaII-mediated cleavage of H-M, the caged initiator I fails to trigger the IR-HCR circuit. Based on a systematic investigation, the IR-HCR circuit readily achieves selective and sensitive analysis of M.SssI MTase and its inhibitors. As a general MTase-sensing platform, the IR-HCR principle was further applied to analyze another MTase (Dam) by redesigning H-M with the Dam recognition sequence. Overall, the versatile homogeneous MTase sensing platform was achieved via an efficient and robust initiator replication amplification circuit and may have enormous potential for early disease diagnosis.
更多
查看译文
AI 理解论文
溯源树
样例
生成溯源树,研究论文发展脉络
Chat Paper
正在生成论文摘要