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Construction of an Enzyme-Free Initiator-Replicated Hybridization Chain Reaction Circuit for Amplified Methyltransferase Evaluation and Inhibitor Assay
- Source :
- Analytical chemistry. 93(4)
- Publication Year :
- 2021
-
Abstract
- 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 ∼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 HM can be recognized and digested by restriction endonuclease HpaII to release initiator I for stimulating a high FRET signal. While the M.SssI-methylated HM prohibits the HpaII-mediated cleavage of HM, 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 HM 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.
- Subjects :
- Concatemer
HpaII
Antimetabolites
010401 analytical chemistry
Deoxyribozyme
Methyltransferases
010402 general chemistry
01 natural sciences
Combinatorial chemistry
0104 chemical sciences
Analytical Chemistry
Restriction enzyme
chemistry.chemical_compound
Förster resonance energy transfer
chemistry
Recognition sequence
Nucleic acid
Azacitidine
Escherichia coli
Fluorouracil
human activities
Nucleic Acid Amplification Techniques
DNA
Subjects
Details
- ISSN :
- 15206882
- Volume :
- 93
- Issue :
- 4
- Database :
- OpenAIRE
- Journal :
- Analytical chemistry
- Accession number :
- edsair.doi.dedup.....71e13bfd22148d65fb59f1235c450e71