From the Cover: PNAS Plus: Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform.DNA sequencing is a fundamental technology in the biological and medical sciences (1). Advances in sequencing technology have enabled the growth of interest in individualized medicine with the hope of better treating human disease. The cost of genome sequencing has dropped by five orders of magnitude over the last decade but still remains out of reach as a conventional clinical tool (2, 3). Thus, the development of new, high-throughput, accurate, low-cost DNA-sequencing technologies is a high priority. Ensemble sequencing-by-synthesis (SBS) platforms dominate the current landscape. During SBS, a DNA polymerase binds and incorporates a nucleotide analog complementary to the template strand. Depending on the instrumentation, this nucleotide is identified either by its associated label or the appearance of a chemical by-product upon incorporation (4). These platforms take advantage of a high-fidelity polymerase reaction but require amplification and have limited read lengths (5). Recently, single-molecule strategies have been shown to have great potential to achieve long read lengths, which is critical for highly scalable and reliable genomic analysis (6–9). Pacific Biosciences’ SMRT SBS approach has been used for this purpose but has lower throughput and higher cost compared with current second-generation technology (10).Since the first demonstration of single-molecule characterization by a biological nanopore two decades ago (11), interest has grown in using nanopores as sensors for DNA base discrimination. One approach is strand sequencing, in which each base is identified as it moves through an ion-conducting channel, ideally producing a characteristic current blockade event for each base. Progress in nanopore sequencing has been hampered by two physical limitations. First, single-base translocation can be too rapid for detection (1–3 μs per base), and second, structural similarities between bases make them difficult to identify unambiguously (12). Some attempts to address these issues have used enzymes as molecular motors to control single-stranded DNA (ssDNA) translocation speeds but still rely on identifying multiple bases simultaneously (13–15). Other approaches used exonuclease to cleave a single nucleoside-5′-monophosphate that then passes through the pore (16), or modified the pore opening with a cyclodextrin molecule to slow translocation and increase resolution for individual base detection (17, 18). All of these techniques rely on detecting similarly sized natural bases, which produce relatively similar current blockade signatures. Additionally, no strategies for covalently linking a single enzyme to a multimeric nanopore have been published.Recently, we reported a method for SBS with nanopore detection (19, 20). This approach has two distinct features: the use of nucleotides with specific tags to enhance base discrimination and a ternary DNA polymerase complex to hold the tagged nucleotides long enough for tag recognition by the nanopore. As shown in Fig. 1, a single DNA polymerase is coupled to a membrane-embedded nanopore by a short linker. Next, template and four uniquely tagged nucleotides are added to initiate DNA synthesis. During formation of the ternary complex, a polymerase binds to a complementary tagged nucleotide; the tag specific for that nucleotide is then captured in the pore. Each tag is designed to have a different size, mass, or charge, so that they generate characteristic current blockade signatures, uniquely identifying the added base. This system requires a single polymerase coupled to each nanopore to ensure any signal represents sequencing information from only one DNA template at a time. Kumar et al. (19) demonstrated that nucleotides tagged with four different polyethylene glycol (PEG) molecules at the terminal phosphate were good substrates for polymerase and that the tags could generate distinct signals as they translocate through the nanopore. These modifications enlarge the discrimination of the bases by the nanopore relative to the use of the natural nucleotides. We recently expanded upon this work by replacing the four PEG polymers with oligonucleotide-based tags and showed that a DNA polymerase coupled to the nanopore could sequentially add these tagged nucleotides to a growing DNA strand to perform Nanopore-SBS (20). Although this work showcased the promise of this technology, it did not describe in detail how to build a protein construct capable of Nanopore-SBS and did not obtain enough data to develop a statistical framework to uniquely distinguish the tagged nucleotides from each other.Open in a separate windowFig. 1.Principle of single-molecule DNA sequencing by a nanopore using tagged nucleotides. Each of the four nucleotides carries a different polymer tag (green square, A; red oval, T; blue triangle, C; black square, G). During SBS, the complementary nucleotide (T shown here) forms a tight complex with primer/template DNA and the nanopore-coupled polymerase. As the tagged nucleotides are incorporated into the growing DNA template, their tags, attached via the 5′-phosphate, are captured in the pore lumen, which results in a unique current blockade signature (Bottom). At the end of the polymerase catalytic reaction, the tag is released, ending the current blockade, which returns to open-channel reading at this time. For the purpose of illustration, four distinct tag signatures are shown in the order of their sequential capture. A large array of such nanopores could lead to highly parallel, high-throughput DNA sequencing.Here, we describe the design and characterization of a protein construct capable of carrying out Nanopore-SBS (Fig. 1). A porin attached to a single DNA polymerase molecule is inserted into a lipid bilayer formed on an electrode array. The polymerase synthesizes a new DNA strand using four uniquely tagged nucleotides. The DNA polymerase is positioned in such a way that when the ternary complex is formed with the tagged nucleotide, the tag is captured by the nanopore and identified by the resulting current blockade signature. We first describe the construction and purification of an α-hemolysin (αHL) heptamer covalently attached to a single ϕ29 DNA polymerase using the SpyTag/SpyCatcher conjugation approach (21), followed by binding of this conjugate with template DNA and its insertion into a lipid bilayer array. We confirm that this complex is stable and retains adequate pore and polymerase activities. We verify that the tagged nucleotides developed by Fuller et al. (20) can be bound by the polymerase and accurately discriminated by the nanopore. We develop an experimental approach and computational methods to uniquely and specifically distinguish true tagged-nucleotide captures from background and from other tagged nucleotides. We address ways that tagged-nucleotide captures may be misidentified and demonstrate approaches to correct for these. We further show this protein construct can capture tagged nucleotides during template-directed DNA synthesis in the presence of Mn²⁺, demonstrating its utility for Nanopore-SBS.