Rapid nanopore discrimination between single polynucleotide molecules

Abstract

A variety of different DNA polymers were electrophoretically driven through the nanopore of an ␣-hemolysin channel in a lipid bilayer. Single-channel recording of the translocation duration and current flow during traversal of individual polynucleotides yielded a unique pattern of events for each of the several polymers tested. Statistical data derived from this pattern of events demonstrate that in several cases a nanopore can distinguish between polynucleotides of similar length and composition that differ only in sequence. Studies of temperature effects on the translocation process show that translocation duration scales as ϳT ؊2 . A strong correlation exists between the temperature dependence of the event characteristics and the tendency of some polymers to form secondary structure. Because nanopores can rapidly discriminate and characterize unlabeled DNA molecules at low copy number, refinements of the experimental approach demonstrated here could eventually provide a low-cost high-throughput method of analyzing DNA polynucleotides.T he discovery that a voltage gradient can drive single-stranded RNA or DNA molecules through a 2-nm transmembrane channel, or nanopore, has opened up the possibility of detecting and characterizing unlabeled polynucleotide molecules at low copy number by using single-channel recording techniques. Because an extended molecule of DNA or RNA can occupy, and thus block, much of an otherwise open aqueous channel, the passage of a single polynucleotide can be monitored by recording the translocation duration and blockade current (magnitude of the reduced ionic flow through the pore) (1). Studies with RNAs of differing base composition have begun to suggest how nanopores could be used to discriminate between different nucleic acid polymers (2).We now extend these observations and provide evidence that each of several different DNA polymers can be identified by a unique pattern in ''event diagrams,'' which are plots of translocation duration vs. blockade current for an ensemble of events. Patterns for a given polymer can be characterized uniquely by three statistical parameters representing the most probable translocation current, I P , the most probable translocation duration, t P , and the characteristic dispersion of values for individual translocation durations, T . Because each type of polynucleotide we use gives rise to specific values of these three parameters, these parameters' values can be used to discriminate rapidly between different types of polynucleotides in a mixed sample. Temperature markedly affects these parameters in two ways. The translocation duration scales as T Ϫ2 for all polymers tested, where T is temperature in°C. The other two parameters exhibit a strong temperature dependence for only those polymers that are known to have stable secondary structure. Materials and MethodsSingle channels were formed in a horizontal bilayer of diphytanoyl phosphatidylcholine by using the protein ␣-hemolysin from Staphylococcus aureus. The horizontal bilayer was formed across a ...

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