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Label-free fluorescence assay coupled exonuclease reaction and SYBR Green I for the detection of T4 polynucleotide kinase activity
摘要： A sensitive label-free ?uorescence assay for monitoring T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on a coupled l exonuclease cleavage reaction and SYBR Green I. In this assay, a double-stranded DNA (dsDNA) was stained with SYBR Green I and used as a substrate for T4 PNK. After the 50-hydroxyl termini of the dsDNA was phosphorylated by the T4 PNK, the coupled l exonuclease began to digest the dsDNA to form mononucletides and single-stranded DNA (ssDNA). At this moment, the ?uorescence intensity of the SYBR Green I decreased because of less a?nity with ssDNA than dsDNA. The decreasing extent was proportional to the concentration of the T4 PNK. After optimization of the detection conditions, including the concentration of ATP, amount of l exonuclease and reaction time, the activity of T4 PNK was monitored by the ?uorescence measurement, with the limit of detection of 0.11 U mL(cid:2)1 and good linear correlation between 0.25–1.00 U mL(cid:2)1 (R2 ? 0.9896). In this assay, no label was needed for ?uorescence detection. Moreover, the inhibition behaviors of the T4 PNK's inhibitors were evaluated by this assay. The result indicated the potential of using this assay for monitoring of the phosphorylation-related process.
关键词： SYBR Green I,label-free ?uorescence assay,T4 polynucleotide kinase activity,exonuclease reaction
更新于2025-09-19 17:13:59
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Determination of the activity of T4 polynucleotide kinase phosphatase by exploiting the sequence-dependent fluorescence of DNA-templated copper nanoclusters
摘要： A fluorometric method is described for the determination of the activity of the enzyme T4 polynucleotide kinase phosphatase (T4 PNKP). A short 3′-terminus phosphorylated DNA strand is hybridized with a long DNA strand to produce a partially double-stranded DNA (dsDNA) substrate. On addition of T4 PNKP, the substrate is dephosphorylated to generate the long dsDNA, and then the long dsDNA acted as a template for synthesizing copper nanoclusters (CuNCs). The dsDNA-templated CuNCs display fluorescence with excitation/emission peak wavelengths of 340/570 nm. The fluorescence is DNA sequence-dependent. A DNA substrate was designed to enhance fluorescence and to reduce the background in order to improve the sensitivity of the assay. The assay has an analytical range that extends from 0.07 U mL?1 to 15 U mL?1 and a detection limit of 0.06 U mL?1.
关键词： Near-zero background,Label-free,Copper nanoclusters,T4 polynucleotide kinase phosphatase activity,Fluorescent biosensing
更新于2025-09-10 09:29:36
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A TiO2/g-C3N4/CdS Nanocomposite-Based Photoelectrochemical Biosensor for Ultrasensitive Evaluation of T4 Polynucleotide Kinase Activity
摘要： Herein, an efficient photoelectrochemical (PEC) platform was constructed by a co-sensitization strategy with a cascade energy level arrangement for the ultrasensitive evaluation of T4 polynucleotide kinase (T4 PNK). Based on CdSe quantum dots (QDs) with an extremely narrow bandgap, this co-sensitization strategy offered a highly efficient sensitizer with a matching band-edge level of a ternary TiO2/g-C3N4/CdS nanocomposite. In this protocol, the ternary nanocomposite was first prepared to serve as the matrix to construct the PEC sensing platform. On the other hand, a well-designed hairpin DNA1 probe with 5’-hydroxyl termini was specifically phosphorylated by T4 PNK which would be selectively cleaved with lambda exonuclease (λ-Exo) outputting 3’-thiol end ssDNA2. After tagged with CdSe QDs, ssDNA2 was captured by the complementary capture DNA3 on the electrode surface. As a result, CdSe QDs were in close contact with the ternary nanocomposite matrix, leading to an enhanced photocurrent response. Therefore, this proposed PEC platform displayed an analytical performance with a wide linear range from 0.0001 to 0.02 U mL-1 and a low detection limit down to 6.9 × 10-5 U mL-1. Moreover, this ternary nanocomposite-based platform exhibited excellent selectivity, good reproducibility, and remarkable storage stability, which shows the great potential for the T4 PNK detection and inhibitor screening.
关键词： CdSe quantum dots,T4 polynucleotide kinase,ternary nanocomposite,co-sensitization strategy,photoelectrochemical biosensor
更新于2025-09-04 15:30:14