Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification

Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

doi:10.1016/j.bios.2015.05.059

Cited by 30 publications

(17 citation statements)

References 43 publications

(37 reference statements)

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“…The IC 50 (half-maximal inhibitory concentration) curves of ADP and (NH 4 ) 2 SO 4 are shown in Figure . The IC 50 values for ADP and (NH 4 ) 2 SO 4 are 1.5 and 12.6 mM, respectively, which are comparable with the literature reported values. − The result confirms that the paper-based fluorescence assay has the potential to screen PNK inhibitors as well as determine corresponding IC 50 values.…”

Section: Resultssupporting

confidence: 87%

“…The detection limit of PNK is 0.0001 U mL –1 according to the 3σ rule, which is lower than or comparative with those of previous reports (Table S1). − ,, The assay–assay precision coefficient of variation (CV) of the PNK activity detection is below 5.1% in all experimental data sets, indicating that the assay has reasonable reproducibility. In addition, the detection principle is also demonstrated by gel electrophoresis analysis (Figure S6).…”

Section: Resultsmentioning

confidence: 85%

“…The traditional PNK activity assays including radioactive isotope 32 P-labeling, polyacrylamide gel electrophoresis (PAGE) and autoradiography , have several limitations such as time-consuming, high cost, radioactive contamination, and requirement of skillful technicians. To overcome these disadvantages, varieties of other techniques for the determination of PNK have been developed including colorimetric, , fluorescent, − electrochemical, , chemiluminescent, and so on. Nevertheless, the above techniques are mainly based on solution-phase reaction systems and need complicated instruments, which are not practical for using outside laboratory and cannot meet the demands of point-of-care (POC) test.…”

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confidence: 99%

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Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance

et al. 2016

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The phosphorylation of nucleic acid with 5'-OH termini catalyzed by polynucleotide kinase (PNK) involves several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was first immobilized on the surface of aldehyde group modified paper, and BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL and lysate of about 50 cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained.

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 85%

mentioning

confidence: 99%

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Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance

et al. 2016

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“…Leung and co-workers have continued their work in this field with recently reported probes of TDG and of several nucleases. , As with molecular beacon based probes, several signal amplification probes have also been reported based on G-quadruplex formation. Such signal amplification probes have been made for the detection of UDG, APE1, and T4 PNK, which claim detection limits between 10 –3 and 10 –4 U/mL.…”

Section: Dna Binding Stimulated Fluorescence 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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“…Polyacrylamide gel electrophoresis (PAGE) associated with radical isotope P-labeling is the conventional standard method for T4 PNK determination. − However, there are several inevitable drawbacks of this method, such as exposure of the operator to radiation, as well as complicated and time-consuming manipulations. To solve these problems, numerous effective strategies have been developed, including colorimetric methods, , electrochemical methods, − , nanoparticle-based methods, − , and fluorescence methods. ,− ,− , Among them, the fluorescence-mediated strategy has been widely applied in T4 PNK detection due to its high sensitivity, simple manipulations, and easy signal readout. For instance, Song et al initially developed a single-labeled DNA molecular beacon for determination of T4 PNK associated with lambda exonuclease (λ exo) digestion .…”

Section: Introductionmentioning

confidence: 99%

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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Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification

Cited by 30 publications

References 43 publications

Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance

Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance

Fluorescent Probes of DNA Repair

Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform

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