51. Label-free and sensitive fluorescent sensing of ten-eleven translocation enzyme via cascaded recycling signal amplifications.
- Author
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Xiang, Jie, Zhang, Junyi, Liao, Lei, Jiang, Bingying, Yuan, Ruo, and Xiang, Yun
- Subjects
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DIOXYGENASES , *DEOXYRIBOZYMES , *DNA probes , *HAIRPIN (Genetics) , *QUADRUPLEX nucleic acids , *DETECTION limit - Abstract
The sensitive detection of ten-eleven translocation (TET) dioxygenase is of significance for understanding the demethylation mechanism of 5-methylocytosine (5mC), which is responsible for a wide range of biological functions that can affect gene expression in eukaryotic species. Here, a non-label and sensitive fluorescence biosensing method for TET assay using TET1 as the model target molecule is established on the basis of target-triggered Mg2+-dependent DNAzyme and catalytic hairpin assembly (CHA)-mediated multiple signal amplification cascades. 5mC sites in the hairpin DNA probe are first oxidized by TET1 into 5-carboxycytosine, which are further reduced by pyridine borane into dihydrouracil, followed by its recognition and cleavage by the USER enzyme to liberate active DNAzyme and G-quadruplex sequences from the probe. The DNAzyme further cyclically cleaves the substrate hairpins to trigger subsequent CHA reaction and DNAzyme cleavage cycles for yielding many G-quadruplex strands. Thioflavin T dye then intercalates into G-quadruplexes to cause a magnificent increase of fluorescence for high sensitivity assay of TET1 with 47 fM detection limit. And, application of this method for TET1 monitoring in diluted serum has also been confirmed. [Display omitted] • Non-label and sensitive fluorescent ten-eleven translocation enzyme assay is developed. • DNAzyme and CHA recycling are integrated into the assay to amplify the signal output. • The method shows high selectivity and has a low detection limit of 47 fM. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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