Citation: Hu, T.; Ma, C.; Yan, Y.;
Chen, J. Detection of DNA
Methyltransferase Activity via
Fluorescence Resonance Energy
Transfer and Exonuclease-Mediated
Target Recycling. Biosensors 2022, 12,
395. https://doi.org/10.3390/
bios12060395
Received: 7 May 2022
Accepted: 6 June 2022
Published: 8 June 2022
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Article
Detection of DNA Methyltransferase Activity via Fluorescence
Resonance Energy Transfer and Exonuclease-Mediated
Target Recycling
Tingting Hu
1,†
, Changbei Ma
1,†
, Ying Yan
1
and Junxiang Chen
2,
*
1
School of Life Sciences, Central South University, Changsha 410013, China; hutingting516@163.com (T.H.);
macb2012@csu.edu.cn (C.M.); yany2018@csu.edu.cn (Y.Y.)
2
Department of Urology, The Second Xiangya Hospital of Central South University, Changsha 410011, China
* Correspondence: chenjxly@csu.edu.cn
† These authors have contributed equally to this work.
Abstract:
In this study, a sensitive method for detecting DNA methyltransferase (MTase) activity
was developed by combining the effective fluorescence resonance energy transfer (FRET) of cationic
conjugated polymers and exonuclease (Exo) III–mediated signal amplification. DNA adenine MTase
targets the GATC sequence within a substrate and converts the adenine in this sequence into N6-
methyladenine. In the method developed in this study, the methylated substrate is cleaved using
Dpn I, whereby a single-stranded oligodeoxynucleotide (oligo) is released. Afterward, the oligo is
hybridized to the 3
0
protruding end of the F-DNA probe to form a double-stranded DNA, which
is then digested by Exo III. Subsequently, due to weak electrostatic interactions, only a weak FRET
signal is observed. The introduction of the Exo-III–mediated target-recycling reaction improved the
sensitivity for detecting MTase. This detection method was found to be sensitive for MTase detection,
with the lowest detection limit of 0.045 U/mL, and was also suitable for MTase-inhibitor screening,
whereby such inhibitors can be identified for disease treatment.
Keywords:
DNA methyltransferase; cationic conjugated polymers; fluorescence resonance energy
transfer; exonuclease III-assisted signal amplification
1. Introduction
As one of the most important epigenetic modifications, DNA methylation highly
affects gene expression, genomic stability, cell growth, and cell senescence [
1
,
2
]. DNA
methyltransferases (MTases) transfer the methyl group of S-adenosine methionine (SAM)
to the N6 position of adenine, or C5 or N4 position of cytosine, thereby protecting the DNA
substrate from catalysis by restriction enzymes [
3
]. Although the nucleotide sequence does
not change after DNA methylation, the expression of the corresponding gene is affected.
MTases are involved in numerous complex biological events, such as aging, Alzheimer’s
disease, and tumorigenesis [
4
]. DNA-methylation tests show 95% accuracy in detecting
cancers. In addition, such tests can identify 97% and 94% of liver and lung metastases
from colorectal cancer, respectively [
5
]. As a potential biomarker, abnormal MTase activity
leads to a wide range of physiological or pathological changes. Therefore, the development
of a highly sensitive and specific method for the detection of MTase activity is of great
significance and has increasingly attracted attention.
The traditional methods used for detecting MTase activity include gel electrophore-
sis, high-performance liquid chromatography, and radioisotope-labeling strategies [
6
,
7
].
Although these methods are well established, most of them either are labor-intensive or
are involved the use of radioactive materials. Several new methods, such as a colorimetric
method based on G-quadruplex, and one based on DNA-gold nanoparticles with electro-
chemical biosensors [
8
,
9
], have been developed to overcome these shortcomings. However,
Biosensors 2022, 12, 395. https://doi.org/10.3390/bios12060395 https://www.mdpi.com/journal/biosensors
