Quencher-free fluorescence strategy for detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification

Liu, Haisheng; Ma, Changbei; Zhou, Meijuan; Chen, Hanchun; He, Hui; Wang, Kemin

doi:10.1007/s00216-016-9914-0

Cited by 9 publications

(3 citation statements)

References 29 publications

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“…2-Aminopurine (2-AP)—whose fluorescence is strongly quenched when incorporated within DNA—has been used as a fluorescent analogue of adenine for many years [33]. 2-AP is often used as a quencher-free fluorescent probe for the study of protein–DNA interactions and DNA flipping [34,35].…”

Section: Introductionmentioning

confidence: 99%

An Exonuclease I-Based Quencher-Free Fluorescent Method Using DNA Hairpin Probes for Rapid Detection of MicroRNA

Liu

et al. 2017

Sensors

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MicroRNAs (miRNAs) act as biomarkers for the diagnosis of a variety of cancers. Since the currently used methods for miRNA detection have limitations, simple, sensitive, and cost-effective methods for the detection of miRNA are required. This work demonstrates a facile, quencher-free, fluorescence-based analytical method for cost-effective and sensitive detection of miRNA using a super 2-aminopurine (2-AP)-labeled hairpin probe (HP) and exonuclease I activity. Specifically, the fluorescence of 2-AP is strongly quenched when it is incorporated within DNA. In the presence of a target miRNA, HP attains an open conformation by hybridizing with the target miRNA to form a double-stranded structure with a protruding 3′-terminus. Next, the digestion of the protruding 3′-terminus is triggered by exonuclease I, during which 2-AP is released free in solution from the DNA, thereby increasing fluorescence. This method is highly sensitive, with a detection limit of 0.5 nM—10 times lower than a previously reported quencher-free fluorescence method. Furthermore, this method has potential applications in clinical diagnosis and biomedical research.

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Section: Introductionmentioning

confidence: 99%

An Exonuclease I-Based Quencher-Free Fluorescent Method Using DNA Hairpin Probes for Rapid Detection of MicroRNA

Liu

et al. 2017

Sensors

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“…The limit of detection (LOD) of the developed method was estimated at 0.045 U/mL according to the 3σ. This LOD value is even lower than that of electrochemiluminescence methods based on methylene blue [ 9 ], colorimetric methods based on G-quadruplex [ 23 ], fluorescence methods based on a novel methylation-responsive DNAzyme strategy [ 24 ], or a fluorescence method based on 2-aminopurine and thioflavin T [ 25 , 26 ], which was attributed to the improved amplification efficiency. Accordingly, the detection limit of the method developed in this study is comparable or even superior to that of most methods used for MTase detection in recent years ( Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

Detection of DNA Methyltransferase Activity via Fluorescence Resonance Energy Transfer and Exonuclease-Mediated Target Recycling

Yan

et al. 2022

Biosensors

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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 3ʹ 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.

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“…Double stranded DNA with a blunt or recessed 3′ end is the best substrate of EXO III. It gradually removes the single nucleotides along the 3′—hydroxyl terminus to the 5′ end of the double stranded DNA [ 17 ], which has no cleavage function for dsDNA containing a protruded 3′ end or single stranded DNA (ssDNA) [ 18 ]. EXO III–assisted electrical signal amplification has been widely used to develop novel biosensing platforms for nucleic acid detection because there are no special requirements for the sequence of substrates [ 19 ].…”

Section: Introductionmentioning

confidence: 99%

Electrochemical Biosensor for Detection of the CYP2C19*2 Allele Based on Exonuclease Ⅲ

Chen

Liu

et al. 2023

Micromachines

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Currently, the therapeutic effect of clopidogrel differs considerably among individuals and is thought to be closely related to the genetic polymorphism of CYP2C19. The CYP2C19*2 gene can reduce the antiplatelet aggregation effect of clopidogrel, which increases the risk of major cardiovascular adverse events in patients. In this research, we report a new type of biosensor for the highly sensitive detection of the CYP2C19*2 gene based on exonuclease III assisted electric signal amplification and the use of calixarene to enrich electrical signal substances. Specifically, under the best conditions, the logarithmic concentrations of the analytes have a good linear relationship with the peak current in the range of 0.01 fM to 100 pM and the detection limit is 13.49 aM. The results have also shown that this method has good selectivity, high sensitivity, and stability, etc., and will provide a very promising application for the detection of the CYP2C19*2 gene and other biological molecules by replacing corresponding nucleic acid sequences.

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Quencher-free fluorescence strategy for detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification

Cited by 9 publications

References 29 publications

An Exonuclease I-Based Quencher-Free Fluorescent Method Using DNA Hairpin Probes for Rapid Detection of MicroRNA

An Exonuclease I-Based Quencher-Free Fluorescent Method Using DNA Hairpin Probes for Rapid Detection of MicroRNA

Detection of DNA Methyltransferase Activity via Fluorescence Resonance Energy Transfer and Exonuclease-Mediated Target Recycling

Electrochemical Biosensor for Detection of the CYP2C19*2 Allele Based on Exonuclease Ⅲ

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