Nanopore DNA sequencing with MspA

Abstract

Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.

Fig. 1.

Crystal structure of MspA. The cross-sectional view through M1-NNN-MspA’s structure using a space-filling model displays the classes of amino acids: Red are positively charged; blue are negatively charged; purple are polar; yellow are hydrophobic-aliphatic; orange are hydrophobic-aromatic. The single constriction, with dimensions similar to that of a single-nucleotide, makes MspA a good candidate for nanopore DNA sequencing techniques.

Fig. 2.

DNA translocation through the nanopore MspA. The cartoon depicts DNA translocation through MspA and the resulting residual current. (A) The positive voltage attracts the negatively charged hairpin DNA into the pore. (B) The DNA threads through the pore until the wider hairpin duplex prevents further translocation. (C) After a few milliseconds the hairpin dissociates allowing for complete translocation. (D) The resulting current trace associated with the above cartoon shows that the hairpin DNA present in the pore allows a residual current, I_res, until the hairpin duplex dissociates.

Fig. 3.

Example histograms of the averaged residual ion currents, 〈I_res〉 are shown for different “hompolymer” single-stranded tails of a 14 base pair hairpin (hp). Data were taken at (A) 180 mV and (B) 140 mV. The translocations included in the above histograms have durations longer than 1 millisecond and reveal distinguishable and well-resolved current levels. We give the average of the fitted Gaussian mean of a number of experiments in the main text. There were at least four experimental repetitions with each of the above hairpin DNA. The reduction in widths at the 140 mV is due to increased time averaging because the dissociation times are nearly 30 × longer than the dissociation times at 180 mV. Additional information may be found in Table S1.

Fig. 4.

Residual current histograms due to single nucleotide substitutions in an otherwise poly-dA hairpin tail. (A) For comparison purposes the top panel summarizes the averaged Gaussian mean and width of I_res of the homopolymer hairpin tails at 180 mV (with fit values in text). Colors of black, blue, red, and green are used to help separate effects on I_res due to dA, dG, dC, and dT, respectively. The residual current markedly changes with the position, x, of a single nucleotide dN_x, within an otherwise poly-dA homopolymer hairpin (hp) tail. (B) When the nucleotide substitution is adjacent to the double-stranded terminus, x = 1, the residual current deviates to resemble the hompolymer values associated with the substituted nucleotide. The dT₁ substitution most closely resembles I_dT. (C) At x = 2, the nucleotide substitution also causes residual current is closer to the homopolymer associated with the substituted nucleotide. The dC₂ substitution is closest to I_dC. (d) With any substitution at x = 3, I_res is only slightly different fromI_dA, suggesting that MspA is primarily sensitive to the two nt after the hairpin duplex. A dG_x substitution at x = 1, 2, or 3, does not significantly influence the current, as may be expected given the relative closeness of I_dG and I_dA.

Fig. 5.

Demonstration of duplex interrupted (DI) nanopore sequencing using MspA. We use synthesized DNA simulating analyte DNA converted to have duplexes between information carrying nucleotides. Each of these duplexes must be sequentially melted as the DNA is pulled through the pore, enabling the residual current to determine the sequence. We provide proof of principle of this technique using DNA chosen to represent different analyte sequences (A) 3′-ATGC-5′, (B) 3′-TACG-5′, and (C) the “blind” sequence determined to be 3′-GTCAC-5′. We display example traces of residual currents (left). Each step in the residual current is representative of three nucleotides within MspA’s constriction held by a 14 bp DNA duplex. For each of these steps we generate a histogram of each level (right) for N translocations. These results are generated from three or more experiments at 140 mV. At higher voltages, the number of translocations increases (Tables S3, S4, and S5), but the level specificity decreases (Figs. S2, S3, and S4) due to reduced time averaging.