Sensitive and Rapid Screening of T4 Polynucleotide Kinase Activity and Inhibition Based on Coupled Exonuclease Reaction and Graphene Oxide Platform

Abstract

Phosphorylation of DNA with 5'-hydroxyl termini plays a critical role in a majority of normal cellular events, including DNA recombination, DNA replication, and repair of DNA during strand interruption. Determination of nucleotide kinase activity and inhibition is under intense development due to its importance in regulating nucleic acid metabolism. Here, by using T4 polynucleotide kinase (PNK) as a model, which plays an essential role in cellular nucleic acid metabolism, particularly in the cellular responses to DNA damage, we describe a strategy for simply and accurately determining nucleotide kinase activity and inhibition by means of a coupled λ exonuclease cleavage reaction and graphene oxide (GO) based platform. The dye attached dsDNA preserves most of the fluorescence when mixed with GO. While dsDNA is phosphorylated by PNK and then immediately cleaved by λ exonuclease, fluorescence is greatly quenched. Because of the super quenching ability and the high specific surface area of GO, the as-proposed platform presents an excellent performance with wide linear range and low detection limit in the cell extracts environment. Additionally, inhibition effects of adenosine diphosphate, ammonium sulfate, and sodium hydrogen phosphate have also been investigated. The method not only provides a universal platform for monitoring activity and inhibition of nucleotide kinase but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.

Aptasensors Based on Stripping Voltammetry

“…More importantly, aptasensors can be used in a wide variety of sample matrixes, including non-physiological buffers and temperature conditions that would cause denaturation of typical antibodies [12]. Owing to the above advantages, many different aptasensors, such as fluorescent [13], colorimetric [14], electrochemical [4,15], and electrochemiluminescent [16][17][18][19] approaches have been developed to date. It covers nearly all biosensing strategies.…”

Preparation of Fluorescent Graphene Quantum Dots as Biological Imaging Marker for Cells

“…石墨烯因 其良好的导电、导热、力学等物理性能 [3] , 在电池 [4] 、储 氢材料 [5] 、药物传递 [6] 、生物探针与传感 [7,8] 等方面具有 广 泛 的 应 用 [9,10] . 理 论 和 实 验 表 明 , 石 墨 烯 量 子 点 (Graphene Guantum Dots, GQDs)由于边缘效应 [11] 和小尺 寸效应 [12] , 可以表现出独特的光化学性质.…”

Highly specific fluorescence detection of T4 polynucleotide kinase activity via photo-induced electron transfer

“…Liu et al [20] realized the sensitive detection of T4 polynucleotide kinase activity by coupling ligation-triggered DNAzyme cascade and split DNAzyme amplification. Wu et al [18] and Lin et al [21] respectively, realized the detection of T4 PNK activity by using graphene oxide as a super quencher.…”

“…Although these fluorescence strategies have made great advances toward the DNA phosphorylation assay, they always utilize dye double-labeled molecular beacon (MB) [19,20] or single-labeled probes combining with nanomaterials as quenchers [18,21].…”