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首页> 外文会议>NATO Advanced Research Workshop on Biological, Biophysical amp; Theoretical Aspects of Polymer Structure and Transport Jun 20-25, 1999 Bikal, Hungary >USE OF A NANOSCALE PORE TO READ SHORT SEGMENTS WITHIN SINGLE POLYNUCLEOTIDE MOLECULES
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USE OF A NANOSCALE PORE TO READ SHORT SEGMENTS WITHIN SINGLE POLYNUCLEOTIDE MOLECULES

机译：使用纳米孔读取单核苷酸分子内的短链段

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Single-stranded polynucleotide molecules impede ionic current when they are driven through a nanoscale pore formed by the a-hemolysin heptameric channel (see J. Kasianowicz, D. Deamer, D. Branton, this volume). The duration and frequency of these blockades correlate with the length and concentration of the polymer examined. This suggests that translocation of RNA and DNA strands through the nanopore might also be used to derive a direct, high-speed readout of each molecule's linear composition. We have recently shown that this is possible. Homopolymers of polycytidylic acid (poly C), polyadenylic acid (poly A), and polyuridylic acid (poly U) cause blockades of current through the α-hemolysin pore that are distinguishable from one-another based on amplitude and duration. These differences are due to the predominant secondary structure adopted by each homopolymer at room temperature in neutral buffer. We have also demonstrated that the nanopore instrument has sufficient sensitivity and resolution to detect short, discrete blocks within single polynucleotide molecules during translocation. For example, within an individual RNA strand, the transition from a 30-nucleotide poly A segment to a 70-nucleotide poly C segment can be read as an abrupt, 10 pA current change. A larger current change can also be observed at the transition between nucleotides and an abasic deoxyribose-phosphate segment inserted into a synthetic DNA strand. These polymers may be used to encode targeting molecules such as antibodies, gene specific oligonucleotides, and peptide agonists.
机译：当单链多核苷酸分子被驱动通过α-溶血素七聚体通道形成的纳米级孔时，它们会阻碍离子电流（请参见J. Kasianowicz，D. Deamer，D. Branton，此册）。这些封锁的持续时间和频率与所检查的聚合物的长度和浓度相关。这表明RNA和DNA链穿过纳米孔的移位也可用于得出每个分子线性组成的直接，高速读数。我们最近表明，这是可能的。聚胞苷酸（poly C），聚腺苷酸（poly A）和聚尿苷酸（poly U）的均聚物会导致通过α-溶血素孔的电流阻塞，这可以根据振幅和持续时间彼此区分。这些差异是由于每种均聚物在室温下在中性缓冲液中采用的主要二级结构所致。我们还证明了纳米孔仪器具有足够的灵敏度和分辨率，可在移位期间检测单个多核苷酸分子内的短而离散的嵌段。例如，在单个RNA链中，从30个核苷酸的poly A片段到70个核苷酸的poly C片段的转变可以理解为突然的10 pA电流变化。在核苷酸和插入合成DNA链的脱氧核糖磷酸片段之间的过渡处，也可以观察到更大的电流变化。这些聚合物可用于编码靶向分子，例如抗体，基因特异性寡核苷酸和肽激动剂。

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