Use of a Nanoscale Pore to Read Short Segments within Single Polynucleotide Molecules

Abstract

Single-stranded polynucleotide molecules impede ionic current when they are driven through a nanoscale pore formed by the α-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.