title: Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array.
abstract: 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.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115.
authors.forename: P Benjamin
authors.initials: PB
authors.lastname: Stranges
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115.|Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA 02115.
authors.forename: Mirkó
authors.initials: M
authors.lastname: Palla
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.
authors.forename: Sergey
authors.initials: S
authors.lastname: Kalachikov
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115.
authors.forename: Jeff
authors.initials: J
authors.lastname: Nivala
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Michael
authors.initials: M
authors.lastname: Dorwart
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Andrew
authors.initials: A
authors.lastname: Trans
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.
authors.forename: Shiv
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.
authors.forename: Mintu
authors.initials: M
authors.lastname: Porel
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.
authors.forename: Minchen
authors.initials: M
authors.lastname: Chien
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.
authors.forename: Chuanjuan
authors.initials: C
authors.lastname: Tao
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.
authors.forename: Irina
authors.initials: I
authors.lastname: Morozova
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.
authors.forename: Zengmin
authors.initials: Z
authors.lastname: Li
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.
authors.forename: Shundi
authors.initials: S
authors.lastname: Shi
authors.affiliation: Department of Biomedical Engineering, Arizona State University, Tempe, AZ 85281.
authors.forename: Aman
authors.initials: A
authors.lastname: Aberra
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Cleoma
authors.initials: C
authors.lastname: Arnold
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Alexander
authors.initials: A
authors.lastname: Yang
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Anne
authors.initials: A
authors.lastname: Aguirre
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Harada
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Daniel
authors.initials: D
authors.lastname: Korenblum
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: James
authors.initials: J
authors.lastname: Pollard
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Ashwini
authors.initials: A
authors.lastname: Bhat
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Gremyachinskiy
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Arek
authors.initials: A
authors.lastname: Bibillo
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Roger
authors.initials: R
authors.lastname: Chen
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Randy
authors.initials: R
authors.lastname: Davis
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.
authors.forename: James J
authors.initials: JJ
authors.lastname: Russo
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.|Genia Technologies, Santa Clara, CA 95050.
authors.forename: Carl W
authors.initials: CW
authors.lastname: Fuller
authors.affiliation: Genia Technologies, Santa Clara, CA 95050.
authors.forename: Stefan
authors.initials: S
authors.lastname: Roever
authors.affiliation: Center for Genome Technology and Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027.|Columbia University College of Physicians and Surgeons, New York, NY 10032.
authors.forename: Jingyue
authors.initials: J
authors.lastname: Ju
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115; gchurch@genetics.med.harvard.edu.|Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA 02115.|Genia Technologies, Santa Clara, CA 95050.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase; D004566:Electrodes; D004867:Equipment Design; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation* / Q000379:methods*; D008958:Models, Molecular; D058608:Nanopores; D009711:Nucleotides / Q000032:analysis / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D018272:Porins / Q000378:metabolism
chemical_list: D009711:Nucleotides; D011108:Polymers; D018272:Porins; D004259:DNA-Directed DNA Polymerase
keywords: integrated electrode array; nanopore sequencing; polymer-tagged nucleotides; protein design; single-molecule detection