Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore
Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore
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DOI：
10.1038/nnano.2007.344
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发表时间：
2007-11-01
影响因子：
38.3
通讯作者：
Akeson, Mark
中科院分区：
文献类型：
--
作者：
Benner, Seico;Chen, Roger J. A.;Akeson, Mark
纳米孔具有用作生物传感器的潜力，并且是分析单个DNA或RNA分子结构和组成的成熟工具（1-3）。最近，纳米孔已被用于测量酶与其DNA底物的结合（4,5）。在这种技术中，通过施加电压将与酶结合的多核苷酸吸入纳米孔中。电场作用在多核苷酸带电主链上的力被用来研究酶与多核苷酸的相互作用。在这里，我们展示了一个纳米孔传感器可以准确地识别DNA模板结合在单个DNA聚合酶分子的催化位点。利用有限状态机逻辑实现了对未结合DNA、二元DNA/聚合酶复合物和三元DNA/聚合酶/脱氧核苷酸三磷酸复合物的实时区分。这项技术适用于许多结合或修饰DNA或RNA的酶，包括外切酶、激酶和其他聚合酶。
Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules(1-3). Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates(4,5). In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/ polymerase/ deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.