Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase

Abstract

Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction 1,2 . Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) 3 with phi29 DNA polymerase (DNAP) 4 , which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ~28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With singlenucleotide resolution and DNA translocation control, this system integrates solutions to two longstanding hurdles to nanopore sequencing 2 .In nanopore DNA sequencing, a pore inserted into a membrane permits the flow of ionic current when a voltage is applied across the membrane. As a strand of DNA passes through the pore, it causes changes in current that can be related to the sequence of the DNA. Such a strategy offers the promise of rapidly sequencing long single molecules of DNA 2 , amplification-free sample preparation 1 and direct detection of epigenetic modifications such as base methylation 3,5 . Recent progress toward nanopore sequencing has included determining the resolution and characterizing the recognition sites of biological nanopores MspA 3,6 and α-hemolysin 7-9 as well as developing a method to slow DNA translocation though a nanopore 4,6,10 . However, to our knowledge no system has yet been reported that can read nucleotide-specific current levels as an unmodified strand of DNA passes through a nanopore. In this work, we read DNA by detecting current levels associated with singlenucleotide movement of the strand through MspA. Base-calling algorithms will need to be developed to translate these ionic current reads into a DNA sequence.

Graphene nanodevices for DNA sequencing

“…Recently, a viscosity gradient, involving an ionic liquid BmimPF6 on the cis side and a 2M KCl solution on the trans side, was used to lower the DNA translocation speed by two orders of magnitude [121]. A very different approach is to employ a polymerase or helicase enzyme to open the dsDNA helix and slowly ratchet one of its strands through the pore channel [27,28].…”

“…Nucleic acid translocations through α-haemolysin pores in lipid membranes were measured already nearly two decades ago [26], motivated by the idea to read the consecutive bases of a ss-DNA molecules in linear fashion. Since these early days, the nanopore field has grown tremendously, and excitingly, DNA sequencing with nanopores has indeed been realised [27,28]. Solid-state nanopores present some interesting advantages over their biological counterparts, such as high stability, control over pore diameter and channel length, lower sensitivity to external parameters such as pH, temperature, salt concentration and mechanical stress, and, importantly, they are well suited for massive upscaling and device integration on chip [29].…”

Dendrimers in Nanoscale Confinement: The Interplay between Conformational Change and Nanopore Entrance

“…Biological nanopores help control molecular traffic across cellular membranes, whereas natural and man-made pores are exploited in nanotechnology for filtration 3 as well as the label-free sensing of analytes 4−8 including electrical sequencing of individual DNA strands. 9,10 Confining dendrimers temporary or permanently into nanopores is of relevance in basic and applied science. In research, permeating dendrimers of different size are used to probe the lumen of biological and inorganic nanopores, 11 whereas in biomedicine, the interaction influences the transport of encapsulated therapeutics across biological membranes 12,13 or achieves the blocking of pore-forming toxins.…”

The emergence of nanopores in next-generation sequencing

“…Recent advances, such as the shorter and narrower nanopore MspA, permit the identification of four nucleotides residing inside the nanopore by determining the amount of blocked current ( figure 3(b)). Combining the MspA with the phi29 DNA polymerase can slow the translocation of DNA through the nanopore and subsequently sequence 50-nt-long DNA fragments ( figure 3(a)) [19]. Recent advances in signal processing have extended this approach to 5-kilobase-long DNA strands [20].…”