Detecting SNPs Using a Synthetic Nanopore

Abstract

We have discovered a voltage threshold for permeation through a synthetic nanopore of dsDNA bound to a restriction enzyme that depends on the sequence. Molecular Dynamic simulations reveal that the threshold is associated with a nanoNewton force required to rupture the DNA-protein complex. A single mutation in the recognition site for the restriction enzyme, i.e. a single nucleotide polymorphism (SNP), can easily be detected as a change in the threshold voltage. Consequently, by measuring the threshold voltage in a synthetic nanopore, it may be possible to discriminate between two variants of the same gene (alleles) that differ in one base.Restriction enzymes are used prevalently in recombinant DNA technology for cleaving doublehelical DNA segments containing a specific target sequence. Another use is genotyping. Because the binding to the target is extraordinarily sequence specific, restriction enzymes can be used to identify single nucleotide polymorphisms (SNPs) that occur when variants of the same gene (alleles) differ in one base.We have discovered a method for discriminating between alleles that uses a synthetic nanopore to measure the binding of a restriction enzyme to DNA. When a voltage is applied across a membrane containing a nanopore, polyanionic DNA immersed in electrolyte at the cathode diffuses toward the anode and is driven across the membrane by the electric field in the pore. The force due to the field acting on the strand during the translocation impels DNA to bend and stretch within the pore. 1-4 At low fields ℰ < 500mV/10nm, double-stranded DNA (dsDNA) easily permeates pores with diameters ≥2.4nm because the double helix (~2nm diameter) is smaller than the pore. 5 But the permeability of DNA through the pore changes dramatically if it is bound to a restriction enzyme.To study the binding of a restriction enzyme like EcoRI to DNA, we introduced an excess of the enzyme in solution with DNA without the Mg +2 cofactor that is required for cleaving the nucleic acid. Under these conditions, EcoRI is thought to bind and diffuse along DNA. 6,7 The diffusive motion along the strand is arrested at the cognate site, i.e. -GAATTC-for EcoRI. Bulk measurements of the binding at the cognate site indicate a free energy of formation ΔG =−15.2kcal/mol. 6-9 However, the introduction of any mutation among the cognate sites produces a position-dependent reduction in the binding energy that ranges from 6-13kcal/mol. 8,9 Site-specific DNA-binding proteins also have an affinity for nonspecific DNA. In contrast with site-specific binding or binding to a non-cognate site with a single nucleotide mutation, a nonspecifically bound complex is not localized to a particular site. For EcoRI, sites that differ from the cognate sequence by two or more base-pairs(bps) are considered nonspecific since they are not cleaved and show low binding constants. For a nonspecifically bound EcoRI-DNA complex, the free energy of formation is reduced to −4.8kcal/mol. 8,9We measured the permeability of dsDNA in solution with EcoRI ...

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