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A sensitive immobilization-free electrochemical assay for T4PNK activity based on exonuclease III-assisted recycling

机译：基于外切核酸酶III辅助再循环的T4PNK活性的无敏固化电化学测定

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摘要

Coupling the properties of T4 polynucleotide kinase (T4PNK) catalyzing the transfer of ATP gamma-phosphate residue to 5'-hydroxyl termini of nucleic acids, with lambda exonuclease (lambda-exo), a highly processive 5'-3' exonuclease, digesting the 5'-phosphorylated strand of a double DNA to produce single-strand DNA and mononucleotides, this work develops a highly sensitive immobilization-free electrochemical method for the detection of T4PNK activity based on lambda-exo and exonuclease III-assisted signal amplification. Upon the reaction of T4PNK and lambda-exo on substrate-DNA, single-stranded DNA segments (released-ssDNA) are dissociated to hybridize with a methylene blue-labeled hairpin probe (MB-DNA) and then the digestion of MB-DNA from the blunt 3 terminus by exonuclease III is activated, resulting in the release of MB-labeled mononucleotides and the complementary DNA segment, followed by the latter hybridizing with another MB-DNA to initiate the cycling process. With a smaller size and less negative charge, the MB-labeled mononucleotide thus diffuses facilely to the negatively charged indium tin oxide (ITO) electrode, actuating an amplified electrochemical signal, and the detection limit of the proposed assay can reach as low as 0.005 U mL(-1). Additionally, this assay can avoid the sophisticated probe immobilization processes. Therefore, this strategy exhibits the merits of high sensitivity, simplicity, and being immobilization-free for electrochemical assay of T4PNK activity, which is consequently believed to bear considerable potential as a detection platform for related researches.
机译：偶联T4多核苷酸激酶（T4PNK）的性质催化ATPγ-磷酸残基转移至5'-羟基末端的核酸，用λ外切核酸酶（Lambda-EXO），一种高度加工的5'-3'外切核酸酶，消化5'-磷酸化的双DNA链，以产生单链DNA和单核苷酸，这项工作产生了高敏感的固定性电化学方法，用于检测基于Lambda-EXO和Exonuclease III辅助信号扩增的T4PNK活性。在T4PNK和Lambda-exo对基质-DNA上的反应后，将单链DNA段（释放-SSDNA）与亚甲基蓝标记的发夹探针（MB-DNA）杂交，然后从MB-DNA的消化中杂交通过外切核酸酶III钝化3末端被激活，导致MB标记的单核苷酸和互补DNA区段的释放，然后用另一种MB-DNA杂交以引发循环过程。具有较小的尺寸和较少的负电荷，因此Mb标记的单核苷酸因此伸直散射到带负电的氧化铟锡（ITO）电极，致动放大的电化学信号，并且所提出的测定的检测限达到0.005° ml（-1）。另外，该测定可以避免复杂的探针固定过程。因此，该策略表现出高灵敏度，简单性，并且不含T4PNK活性的电化学测定的优点，因此被认为是与相关研究的检测平台承担相当大的潜力。

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  • 来源
    《RSC Advances》 |2015年第92期|共6页
  • 作者

    Wang Yonghong; Wu Yaohui; Wang Yuanqing; Zhou Bo; Wu Shun;

  • 作者单位

    Cent South Univ Forestry &

    Technol Coll Life Sci &

    Technol Changsha 410004 Hunan Peoples R China;

    Cent South Univ Forestry &

    Technol Coll Life Sci &

    Technol Changsha 410004 Hunan Peoples R China;

    Cent South Univ Forestry &

    Technol Coll Life Sci &

    Technol Changsha 410004 Hunan Peoples R China;

    Cent South Univ Forestry &

    Technol Coll Life Sci &

    Technol Changsha 410004 Hunan Peoples R China;

    Cent South Univ Forestry &

    Technol Coll Life Sci &

    Technol Changsha 410004 Hunan Peoples R China;

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  • 正文语种 eng
  • 中图分类 化学;
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