Roger Chen

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

Significance Efficient cost-effective single-molecule sequencing platforms will facilitate deciphering complete genome sequences, determining haplotypes, and identifying alternatively spliced mRNAs. We demonstrate a single-molecule nanopore-based sequencing by synthesis approach that accurately distinguishes four DNA bases by electronically detecting and differentiating four different polymer tags attached to the terminal phosphate of the nucleotides during their incorporation into a growing DNA strand in the polymerase reaction. With nanopore detection, the distinct polymer tags are much easier to differentiate than natural nucleotides. After tag release, growing DNA chains consist of natural nucleotides allowing long reads. Sequencing is realized on an electronic chip containing an array of independently addressable electrodes, each with a single polymerase–nanopore complex, potentially offering the high throughput required for precision medicine. DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

Significance DNA sequencing has been dramatically expanding its scope in basic life science research and clinical medicine. Recently, a set of polymer-tagged nucleotides were shown to be viable substrates for replication and electronically detectable in a nanopore. Here, we describe the design and characterization of a DNA polymerase–nanopore protein construct on an integrated chip. This system incorporates all four tagged nucleotides and distinguishes single–tagged-nucleotide addition in real time. Coupling protein catalysis and nanopore-based detection to an electrode array could provide the foundation of a highly scalable, single-molecule, electronic DNA-sequencing platform. 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.