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A Dual-Enzyme-Assisted Three-Dimensional DNA Walking Machine Using T4 Polynucleotide Kinase as Activators and Application in Polynucleotide Kinase Assays
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-02-08 00:00:00 , DOI: 10.1021/acs.analchem.7b04924
Chang Feng 1 , Zihan Wang 2 , Tianshu Chen 2 , Xiaoxia Chen 2 , Dongsheng Mao 2 , Jing Zhao 2 , Genxi Li 1, 2
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-02-08 00:00:00 , DOI: 10.1021/acs.analchem.7b04924
Chang Feng 1 , Zihan Wang 2 , Tianshu Chen 2 , Xiaoxia Chen 2 , Dongsheng Mao 2 , Jing Zhao 2 , Genxi Li 1, 2
Affiliation
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T4 polynucleotide kinase (T4 PNK), an intracellular kinase, catalyzes the phosphorylation of 5′-hydroxyl termini in nucleic acids and plays a crucial role in DNA-related physiological activities. Malfunctioning of PNK is associated with the deregulation of many cellular activities and eventually induces a variety of human diseases. Herein, we report a smart three-dimensional (3D) DNA walking machine using PNK as an effective activator when coupled with the duplex DNA nuclease-assisted cleavage reaction. The 3D DNA tracks benefit from high DNA loading capacity of gold nanoparticles, and the high efficiency of duplex nuclease-mediated cyclic cleavage facilitates the movement of the DNA machine in response to T4 PNK. The DNA machine is also applied for the PNK assay based on the signal amplification from point to area during the DNA walking process. The method achieves an excellent detection limit of 0.0067 U/mL with a linear range from 0.01 to 0.3 U/mL and a favorable specificity even in complex serum samples. Therefore, the 3D DNA machine shows great potential in biochemical and molecular biology studies, drug discovery, and clinic diagnostics.
中文翻译：
T4多核苷酸激酶为激活剂的双酶辅助三维DNA步行机及其在多核苷酸激酶测定中的应用
T4多核苷酸激酶（T4 PNK）是一种细胞内激酶，可催化核酸中5'-羟基末端的磷酸化，并在与DNA有关的生理活动中起关键作用。PNK的功能异常与许多细胞活性的失调有关，并最终诱发多种人类疾病。在这里，我们报告了一个智能的三维（3D）DNA步行机，当与双工DNA核酸酶辅助的裂解反应结合时，使用PNK作为有效的激活剂。3D DNA轨道受益于金纳米颗粒的高DNA负载能力，并且双链核酸酶介导的环状切割的高效率促进了DNA机器响应T4 PNK的运动。基于DNA步移过程中从点到面的信号放大，DNA机也可用于PNK分析。该方法实现了0.0067 U / mL的极佳检测限，线性范围为0.01至0.3 U / mL，即使在复杂的血清样品中也具有良好的特异性。因此，这种3D DNA机器在生化和分子生物学研究，药物发现和临床诊断中显示出巨大的潜力。
更新日期：2018-02-08
中文翻译：
T4多核苷酸激酶为激活剂的双酶辅助三维DNA步行机及其在多核苷酸激酶测定中的应用
T4多核苷酸激酶（T4 PNK）是一种细胞内激酶，可催化核酸中5'-羟基末端的磷酸化，并在与DNA有关的生理活动中起关键作用。PNK的功能异常与许多细胞活性的失调有关，并最终诱发多种人类疾病。在这里，我们报告了一个智能的三维（3D）DNA步行机，当与双工DNA核酸酶辅助的裂解反应结合时，使用PNK作为有效的激活剂。3D DNA轨道受益于金纳米颗粒的高DNA负载能力，并且双链核酸酶介导的环状切割的高效率促进了DNA机器响应T4 PNK的运动。基于DNA步移过程中从点到面的信号放大，DNA机也可用于PNK分析。该方法实现了0.0067 U / mL的极佳检测限，线性范围为0.01至0.3 U / mL，即使在复杂的血清样品中也具有良好的特异性。因此，这种3D DNA机器在生化和分子生物学研究，药物发现和临床诊断中显示出巨大的潜力。
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