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

Abstract

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a singlemolecule 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 oligonucleotidebased 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 singlemolecule 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.single-molecule sequencing | nanopore | DNA sequencing by synthesis | polymer-tagged nucleotides | chip array T he importance of DNA sequencing has increased dramatically from its inception four decades ago. It is recognized as a crucial technology for most areas of biology and medicine and as the underpinning for the new paradigm of personalized and precision medicine. Information on individuals' genomes and epigenomes can help reveal their propensity for disease, clinical prognosis, and response to therapeutics, but routine application of genome sequencing in medicine will require comprehensive data delivered in a timely and cost-effective manner (1). Although 35 years of technological advances have improved sequence throughput and have reduced costs exponentially, genome analysis still takes several days and thousands of dollars to complete (1, 2). To realize the potential of personalized medicine fully, the speed and cost of sequencing must be brought down another order of magnitude while increasing sequencing accuracy and read length. Singlemolecule approaches are thought to be essential to meet these requirements and offer the additional benefit of eliminating amplification bias (3, 4). Although optical methods for singlemolecule sequencing have been achieved and commercialize...

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

“…All four characteristic current levels for the tags and transitions between them can be readily distinguished. The ability to observe all four tagged nucleotides without the presence of noncatalytic divalent cations, which slows tag release, demonstrates greater potential for sequencing speed then previously shown (20). Homopolymer sequences in the template, and repeated, high-frequency tag capture events of the same nucleotide in the raw sequencing reads were considered a single base for sequence alignment.…”

“…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.…”

“…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.…”

Membrane protein-based biosensors

“…As growing the DNA, the tagged nucleotide binds with the polymerase while the tag composed of a specific oligonucleotide is captured in the pore, released by the reaction, and generates the nucleotide specific blocking signal. This research group developed the system on a complementary metal oxide semiconductor (CMOS) chip and demonstrated 264 parallel measurements [32,33]. The commercial device, MinION, released by Oxford Nanopore Technologies has promoted Blocking levels (pink line) were estimated from a raw current trace (grey line, ii, top), and the levels of the pink line were compared with the predicted levels from the reference sequence (black line, ii, bottom).…”

Nanopore Sensing

“…51 More recently, nanopore-based sequencing-by-synthesis (SBS) has been developed. 52 In Table 1, six widely employed biological nanopores are shown along with corresponding critical dimensions and the analytes that have been detected.…”