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Issue 28, 2018
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Single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase

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Abstract

DNA methylation is a predominant epigenetic modification that plays crucial roles in various cellular processes. DNA methyltransferase (MTase) is responsible for DNA methylation, and its dysregulation may induce aberrant methylation patterns that are closely related to cancers. Conventional methods for DNA MTase assay are usually cumbersome and laborious with poor sensitivity. Alternatively, some signal amplification strategies are employed to improve the sensitivity, but they suffer from poor specificity and consequently limited sensitivity due to the nonspecific amplification. Herein, we develop for the first time a new fluorescence method to specifically and sensitively detect DNA MTase activity on the basis of single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification. In the presence of DNA MTase, the hairpin substrate is methylated and cleaved by endonuclease Dpn I, releasing a 24-nt cleavage product. The 24-nt cleavage product may function as a primer and adjacently hybridize with the ligation probes (LP1 and LP2) to form the template (LP1–LP2) for strand displacement amplification (SDA), initiating the single-ribonucleotide repair-mediated cyclic ligation-dependent SDA to produce a large number of reporter probes. The reporter probe can subsequently hybridize with the signal probe that is modified with FAM and BHQ1 to form a stable double-stranded DNA (dsDNA) duplex with a ribonucleotide mismatch. Ribonuclease HII (RNase HII) can excise the single ribonucleotide, resulting in the cyclic cleavage of signal probes and the generation of an enhanced fluorescence signal. Taking advantage of the high specificity of RNase HII-catalyzed single-ribonucleotide excision and the high amplification efficiency of cyclic ligation-dependent SDA, this assay exhibits the highest sensitivity reported so far with a detection limit of 4.8 × 10−6 U mL−1 and a large dynamic range of 5 orders of magnitude. Moreover, this method can be used for the discrimination of Dam MTase from other DNA MTases, the accurate quantification of Dam MTase activity in E. coli cells, and the screening of Dam MTase inhibitors, providing a new paradigm for biomedical research and clinical diagnosis.

Graphical abstract: Single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase

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Supplementary files

Article information


Submitted
18 May 2018
Accepted
17 Jun 2018
First published
18 Jun 2018

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2018,9, 6053-6061
Article type
Edge Article

Single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase

L. Wang, X. Han, C. Li and C. Zhang, Chem. Sci., 2018, 9, 6053
DOI: 10.1039/C8SC02215A

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material and it is not used for commercial purposes.

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    [Original citation] - Published by The Royal Society of Chemistry (RSC) on behalf of the European Society for Photobiology, the European Photochemistry Association, and RSC.
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    [Original citation] - Published by The Royal Society of Chemistry.

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