An entropy-driven signal amplifying strategy for real-time monitoring of DNA methylation process and high-throughput screening of methyltransferase inhibitors

Chen, Siyi; Ma, Huan; Li, Wang; Nie, Zhou; Yao, Shouzhuo

doi:10.1016/j.aca.2017.03.017

Cited by 15 publications

(3 citation statements)

References 34 publications

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“…For instance, Yang et al detected thrombin with a linear detection range from 100 fM to 10 nM and an LOD of only 30 fM 395 by combining toehold-mediated target recycling, hybridization chain reaction, and electrochemical readout. A variety of strand displacement-based sensors were also developed for the assessment of enzyme activity; this is most naturally achieved for DNA-modifying enzymes such as T4 DNA ligase, 361 methyl transferase, 396 uracil-DNA glycosylase (UDG), 397,398 or telomerase. 399 With the use of aptamer recognition combined with DNA strand displacement, also whole cell sensors have been developed.…”

Section: Other Sensor Schemes Based On Toehold-mediated Strand Displa...mentioning

confidence: 99%

Principles and Applications of Nucleic Acid Strand Displacement Reactions

2019

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Dynamic DNA nanotechnology, a subfield of DNA nanotechnology, is concerned with the study and application of nucleic acid strand-displacement reactions. Strand-displacement reactions generally proceed by three-way or four-way branch migration and initially were investigated for their relevance to genetic recombination. Through the use of toeholds, which are single-stranded segments of DNA to which an invader strand can bind to initiate branch migration, the rate with which strand displacement reactions proceed can be varied by more than 6 orders of magnitude. In addition, the use of toeholds enables the construction of enzyme-free DNA reaction networks exhibiting complex dynamical behavior. A demonstration of this was provided in the year 2000, in which strand displacement reactions were employed to drive a DNAbased nanomachine et al. Nature 2000, 406, 605−608). Since then, toeholdmediated strand displacement reactions have been used with ever increasing sophistication and the field of dynamic DNA nanotechnology has grown exponentially. Besides molecular machines, the field has produced enzyme-free catalytic systems, all DNA chemical oscillators and the most complex molecular computers yet devised. Enzyme-free catalytic systems can function as chemical amplifiers and as such have received considerable attention for sensing and detection applications in chemistry and medical diagnostics. Strand-displacement reactions have been combined with other enzymatically driven processes and have also been employed within living cells (Groves, B.; et al. Nat. Nanotechnol. 2015, 11, 287−294). Strand-displacement principles have also been applied in synthetic biology to enable artificial gene regulation and computation in bacteria. Given the enormous progress of dynamic DNA nanotechnology over the past years, the field now seems poised for practical application.

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Section: Other Sensor Schemes Based On Toehold-mediated Strand Displa...mentioning

confidence: 99%

Principles and Applications of Nucleic Acid Strand Displacement Reactions

2019

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“…In view of this, many methods have been developed to detect DNA MTase activity, such as colorimetric methods with double-stranded DNA (dsDNA) probes [ 7 ], electrochemistry with dsDNA probes and hairpin probes [ 8 , 9 , 10 ], fluorescence using dsDNA probes [ 11 , 12 , 13 ], hairpin probes and dumbbell probes [ 14 , 15 , 16 ], chemiluminescent immunoassays with dsDNA probes [ 17 ] and dumbbell probes [ 17 , 18 , 19 , 20 ], and surface-enhanced Raman scattering (SERS) using dsDNA probes [ 21 ] and hairpin probes [ 22 ]. In addition, these probes usually combine with other nucleotide probes to amplify signals, such as hybridization chain reaction (HCR) [ 23 , 24 ], strand displacement amplification(SDA) [ 25 ], and rolling circle amplification (RCA) [ 18 ], to detect DNA MTase activity precisely. However, other DNA-sequence-assisted nucleic acid amplification methods increase the complexity of experimental protocol design and experimental cost.…”

Section: Introductionmentioning

confidence: 99%

Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe

Wang

Han

Zhou³

et al. 2022

Biosensors

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DNA methyltransferases (MTases) can be regarded as biomarkers, as demonstrated by many studies on genetic diseases. Many researchers have developed biosensors to detect the activity of DNA MTases, and nucleic acid amplification, which need other probe assistance, is often used to improve the sensitivity of DNA MTases. However, there is no integrated probe that incorporates substrates and template and primer for detecting DNA MTases activity. Herein, we first designed a padlock probe (PP) to detect DNA MTases, which combines target detection with rolling circle amplification (RCA) without purification or other probe assistance. As the substrate of MTase, the PP was methylated and defended against HpaII, lambda exonuclease, and ExoI cleavage, as well as digestion, by adding MTase and the undestroyed PP started RCA. Thus, the fluorescent signal was capable of being rapidly detected after adding SYBRTM Gold to the RCA products. This method has a detection limit of approximately 0.0404 U/mL, and the linear range was 0.5–110 U/mL for M.SssI. Moreover, complex biological environment assays present prospects for possible application in intricacy environments. In addition, the designed detection system can also screen drugs or inhibitors for MTases.

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“…Fluorescent biosensors combine specific molecular recognition events with fluorescence conversion processes [32, 33]. Fluorescence sensing systems have the advantages of facile operation, real-time detection, high throughput and easy automation [34–36], and have overcome the limitations of the above methods and thus attracted widespread attention [37–40]. Wang et al developed a label-free fluorescence method through ligation-mediated rolling-circle amplification based on specific oxidation of 5-hydroxymethylcytosine to 5-formylcytosine and then conversion into uracil.…”

Section: Introductionmentioning

confidence: 99%

A novel fluorescent biosensor based on dendritic DNA nanostructure in combination with ligase reaction for ultrasensitive detection of DNA methylation

Zhang

Huang

et al. 2019

J Nanobiotechnol

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BackgroundDNA methylation detection is indispensable for the diagnosis and prognosis of various diseases including malignancies. Hence, it is crucial to develop a simple, sensitive, and specific detection strategy.MethodsA novel fluorescent biosensor was developed based on a simple dual signal amplification strategy using functional dendritic DNA nanostructure and signal-enriching polystyrene microbeads in combination with ligase detection reaction (LDR). Dendritic DNA self-assembled from Y-DNA and X-DNA through enzyme-free DNA catalysis of a hairpin structure, which was prevented from unwinding at high temperature by adding psoralen. Then dendritic DNA polymer labeled with fluorescent dye Cy5 was ligated with reporter probe into a conjugate. Avidin-labeled polystyrene microbeads were specifically bound to biotin-labeled capture probe, and hybridized with target sequence and dendritic DNA. LDR was triggered by adding Taq ligase. When methylated cytosine existed, the capture probe and reporter probe labeled with fluorescent dye perfectly matched the target sequence, forming a stable duplex to generate a fluorescence signal. However, after bisulfite treatment, unmethylated cytosine was converted into uracil, resulting in a single base mismatch. No fluorescence signal was detected due to the absence of duplex.ResultsThe obtained dendritic DNA polymer had a large volume. This method was time-saving and low-cost. Under the optimal experimental conditions using avidin-labeled polystyrene microbeads, the fluorescence signal was amplified more obviously, and DNA methylation was quantified ultrasensitively and selectively. The detection range of this sensor was 10−15 to 10−7 M, and the limit of detection reached as low as 0.4 fM. The constructed biosensor was also successfully used to analyze actual samples.ConclusionThis strategy has ultrasensitivity and high specificity for DNA methylation quantification, without requiring complex processes such as PCR and enzymatic digestion, which is thus of great value in tumor diagnosis and biomedical research.

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An entropy-driven signal amplifying strategy for real-time monitoring of DNA methylation process and high-throughput screening of methyltransferase inhibitors

Cited by 15 publications

References 34 publications

Principles and Applications of Nucleic Acid Strand Displacement Reactions

Principles and Applications of Nucleic Acid Strand Displacement Reactions

Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe

A novel fluorescent biosensor based on dendritic DNA nanostructure in combination with ligase reaction for ultrasensitive detection of DNA methylation

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