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A Single-Molecule Nanopore Device Detects DNA Polymerase Activity With Single-Nucleotide Resolution

机译:单分子纳米孔装置可检测具有单核苷酸分辨率的DNA聚合酶活性

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摘要

The ability to monitor DNA polymerase activity with single-nucleotide resolution has been the cornerstone of a number of advanced single-molecule DNA sequencing concepts. Toward this goal, we report the first spatially-resolved observation of DNA polymerase activity with single-base resolution at the single-molecule level. We describe the design and characterization of a single-species supramolecular nanopore device capable of detecting up to nine consecutive DNA polymerase-catalyzed single nucleotide primer extensions with high sensitivity and spatial resolution (≤ 2.4 Å). The device is assembled in a suspended lipid membrane by threading and mechanically capturing a single strand of DNA-PEG copolymer inside an α-hemolysin protein pore. Single nucleotide primer extensions result in successive displacements of the template DNA strand within the protein pore, which can be monitored by the corresponding stepped changes in the ion current flowing through the pore under an applied transmembrane potential. The system described thus represents a promising advance toward nanopore-mediated single-molecule DNA sequencing concept, and in addition might be applicable to studying a number of other biopolymer-protein interactions and dynamics.
机译:以单核苷酸分辨率监测DNA聚合酶活性的能力一直是许多先进的单分子DNA测序概念的基石。为了实现这一目标,我们报告了DNA聚合酶活性的第一个空间分辨观察结果,在单分子水平上具有单碱基分辨率。我们描述了一种单物种超分子纳米孔装置的设计和表征,该装置能够以高灵敏度和空间分辨率(≤2.4Å)检测多达九个连续的DNA聚合酶催化的单核苷酸引物延伸。通过在α-溶血素蛋白孔内穿线并机械捕获DNA-PEG共聚物的单链,将装置组装在悬浮的脂质膜中。单核苷酸引物的延伸导致模板DNA链在蛋白质孔内的连续移位,这可以通过在施加的跨膜电势下流过孔的离子电流的相应阶梯式变化进行监控。因此,所描述的系统代表了朝纳米孔介导的单分子DNA测序概念发展的有前途的进展,此外还可以用于研究许多其他生物聚合物-蛋白质相互作用和动力学。

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