State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People's Republic of China; School of Pharmacy, Nanjing Medical University, Nanjing 211166, People's Republic of China.
State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People's Republic of China.
Talanta. 2018 Apr 1;180:271-276. doi: 10.1016/j.talanta.2017.12.038. Epub 2017 Dec 14.
5'-Polynucleotide kinase (PNK) is a crucial enzyme that catalyzes the phosphorylation of nucleic acid with 5'-OH termini and this phosphorylation reaction has been involved in many important cellular activities. The evaluation of PNK activity has received an increasing attention due to the significance of PNK. Here, the polydopamine nanospheres (PDANS) could adsorb single-stranded DNA (ssDNA) through π-π stacking or hydrogen bonding between nucleobases and aromatic groups of PDANS, while the interaction between double-stranded DNA (dsDNA) with PDANS was weakened due to the changed conformation. Hence, a novel DNA/PDANS platform was constructed for the sensitive and selective determination of T4 PNK activity based on the preferential binding properties of PDANS for ssDNA over dsDNA and the excellent fluorescence quenching property of PDANS. The dye-labeled dsDNA was phosphorylated by T4 PNK and then digested by λ exonuclease, yielding dye-labeled ssDNA, which would be adsorbed on the surface of the PDANS and the fluorescence was greatly quenched by PDANS. Because of the preferential binding properties of PDANS for ssDNA over dsDNA and the high quenching property of PDANS, the developed DNA/PDANS platform exhibited good analytical performance for T4 PNK sensing in complex biological matrix and applied to screening inhibitors. The proposed DNA/PDANS based platform is promising in developing high-throughput assays for drug screening and clinical diagnostics.
5'-多核苷酸激酶（PNK）是一种关键的酶，可催化 5'-OH 末端的核酸磷酸化，该磷酸化反应参与了许多重要的细胞活动。由于 PNK 的重要性，PNK 活性的评估受到了越来越多的关注。在这里，聚多巴胺纳米球（PDANS）可以通过π-π 堆积或碱基与 PDANS 芳环之间的氢键吸附单链 DNA（ssDNA），而双链 DNA（dsDNA）与 PDANS 之间的相互作用由于构象的改变而减弱。因此，构建了一种新型的 DNA/PDANS 平台，用于基于 PDANS 对 ssDNA 与 dsDNA 的优先结合特性以及 PDANS 优异的荧光猝灭特性，灵敏且选择性地测定 T4 PNK 活性。T4 PNK 使染料标记的 dsDNA 磷酸化，然后用 λ 核酸外切酶消化，得到染料标记的 ssDNA，其将被吸附在 PDANS 的表面上，PDANS 会极大地猝灭其荧光。由于 PDANS 对 ssDNA 与 dsDNA 的优先结合特性以及 PDANS 的高猝灭特性，所开发的 DNA/PDANS 平台在复杂生物基质中对 T4 PNK 检测具有良好的分析性能，并可用于筛选抑制剂。基于 DNA/PDANS 的平台有望用于开发高通量药物筛选和临床诊断的测定方法。
