Quencher-free hairpin probes for real-time detection of T4 polynucleotide kinase activity

Ma, Changbei; Liu, Haisheng; Du, Junyan; Chen, Hanchun; He, Hui; Jin, Shunxin; Wang, Kemin; Wang, Jun

doi:10.1016/j.ab.2015.10.007

Cited by 10 publications

(2 citation statements)

References 26 publications

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“…The excellent performance of this method in real biological samples is guaranteed by the combination of the allosteric effect of the AAP and the enrichment of the fluorescence signal on SA beads. Therefore, we obtained a good detection limit of 0.01 U/mL in buffer and 0.05 U/mL in cell lysates, which is comparable or superior to those of the latest unamplified detection methods. ,,− Finally, this method can be used to estimate the inhibition conditions of various inhibitors, making the AAP an ideal platform for phosphorylation studies and inhibitor screening. To some extent, this method also manifests good versatility as long as changing the design of the target-recognition region.…”

Section: Discussionmentioning

confidence: 70%

Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform

Gao

Guo

Song

et al. 2017

ACS Appl. Mater. Interfaces

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As a vital enzyme in DNA phosphorylation and restoration, T4 polynucleotide kinase (T4 PNK) has aroused great interest in recent years. Therefore, numerous strategies have been established for highly sensitive detection of T4 PNK based on diverse signal amplification techniques. However, they often need sophisticated design, a variety of auxiliary reagents and enzymes, or cumbersome manipulations. We have designed a new kind of allosteric aptamer probe (AAP) consisting of streptavidin (SA) aptamer and the complementary DNA (cDNA) for simple detection of T4 PNK without signal amplification and with minimized interference in complex biological samples. When the 5'-terminus of the cDNA is phosphorylated by T4 PNK, the cDNA is degraded by lambda exonuclease to release the fluorescein amidite (FAM)-labeled SA aptamer, which subsequently binds to streptavidin beads. The enhancement of the fluorescence signal on SA beads can be detected precisely and easily by a microscope or flow cytometer. Our method performs well in complex biological samples as a result of the enrichment of the signaling molecules on beads, as well as simple manipulations to discard the background interference and nonbinding molecules. Without signal amplification techniques, our AAP method not only avoids complicated manipulations but also decreases the time required. With the advantages of ease of operation, reliability, and robustness for T4 PNK detection in buffer as well as real biological samples, the AAP has great potential for clinical diagnostics, inhibitor screening, and drug discovery.

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Section: Discussionmentioning

confidence: 70%

Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform

Gao

Guo

Song

et al. 2017

ACS Appl. Mater. Interfaces

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“…The liberation of the fluorophore from the duplex DNA creates a fluorescent signal (Figure c). Several fluorophore liberation probes of T4 PNK have been reported, all of which function on the basis of 5′ phosphorylation-dependent exonuclease digestion. − Similar to molecular beacon probes, several probes have been developed which rely on strand scission to destabilize a duplex. For example, a probe of AGT has been reported which possesses a methylated restriction site.…”

Section: Naturally Quenched Probesmentioning

confidence: 99%

Fluorescent Probes of DNA Repair

Wilson

Kool

2017

ACS Chem. Biol.

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DNA repair is now understood to play a key role in a variety of disease states, most notably cancer. Tools for studying DNA have typically relied on traditional biochemical methods which are often laborious and indirect. Efforts to study the biology and therapeutic relevance of DNA repair pathways can be limited by such methods. Recently, specific fluorescent probes have been developed to aid in the study of DNA repair. Fluorescent probes offer the advantage of being able to directly assay for DNA repair activity in a simple, mix-and-measure format. This review will summarize the distinct classes of probe designs and their potential utility in varied research and preclinical settings.

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

Liu

Zhou

et al. 2016

Anal Bioanal Chem

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This work demonstrates a novel method for DNA methyltransferase (MTase) activity detection with a quencher-free molecular beacon (MB) probe based on exonuclease (Exo) III-assisted signal amplification. In the presence of Dam MTase and DpnI endonuclease, the elaborately designed hairpin substrate (MB1) was cleaved into two parts (part A and part B). Exo III can then digest part A and release a single-stranded target of the 2-aminopurine-labeled MB (MB2). Subsequently, the MB2 can hybridize with its target to form a double-stranded structure with a protruding 3'-terminus and then trigger the digestion of MB2 by Exo III. During the digestion of MB2, the 2-aminopurine is separated from the DNA strands and released free in solution, inducing an increase of the fluorescent signal. Owing to the presence of a recessed 3'-terminus in the formed double-stranded DNA, Exo III-assisted recyclable cleavage of MB2 was achieved. Therefore, an amplified fluorescence signal was observed. Under the optimized conditions, Dam MTase can be detected in the range of 0.2-40 units/mL with a limit of detection of 0.2 units/mL and good selectivity. Furthermore, the present assay can be used for screening potential DNA MTase inhibitors. Graphical Abstract A quencher-free fluorescence assay for sensitive detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification is reported.

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Quencher-free hairpin probes for real-time detection of T4 polynucleotide kinase activity

Cited by 10 publications

References 26 publications

Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform

Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform

Fluorescent Probes of DNA Repair

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

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