Processive Replication of Single DNA Molecules in a Nanopore Catalyzed by phi29 DNA Polymerase

Abstract

Coupling nucleic acid processing enzymes to nanoscale pores allows controlled movement of individual DNA or RNA strands that is reported as an ionic current time series. Hundreds of individual enzyme complexes can be examined in single-file order at high bandwidth and spatial resolution. The bacteriophage phi29 DNA polymerase (phi29 DNAP) is an attractive candidate for this technology, due to its remarkable processivity and high affinity for DNA substrates. Here we show that phi29 DNAP-DNA complexes are stable when captured in an electric field across the α-hemolysin nanopore. DNA substrates were activated for replication at the nanopore orifice by exploiting the 3′-5′ exonuclease activity of wild-type phi29 DNAP to excise a 3′-H terminal residue, yielding a primer strand 3′-OH. In the presence of deoxynucleoside triphosphates, DNA synthesis was initiated, allowing real time detection of numerous sequential nucleotide additions that was limited only by DNA template length. Translocation of phi29 DNAP along DNA substrates was observed in real time at Angstrom scale precision as the template strand was drawn through the nanopore lumen during replication.Single molecule techniques are now used routinely to study nucleic acids in basic science 1 -3 and technology 4 ,5 . Methods using nanoscale pores (nanopores) are advantageous because they can report the length, structure and composition of unmodified DNA or RNA molecules that are captured in single file order 6-9. Data are typically reported as a time series of ionic current as each DNA strand is driven by an applied electric field across a single pore controlled by a voltage-clamped amplifier. Hundreds to thousands of molecules can be examined at high bandwidth and spatial resolution.Recently, the properties of DNA or RNA molecules bound to nucleic acid processing enzymes have been analyzed at a nanopore orifice. The complexes studied include those of single-stranded DNA with Escherichia coli Exonuclease I 10 , RNA with the bacteriophage phi8 ATPase, 11 , and primer/template DNA substrates bound to the 3′-5′-exonuclease deficient versions of two A-family DNA polymerases, the Klenow fragment of E. coli DNA polymerase (KF(exo-)) and bacteriophage T7 DNA polymerase (T7DNAP(exo-)) 12 -16. We have demonstrated that T7DNAP(exo-) could replicate and advance a DNA template held in the α-hemolysin (α-HL) nanopore against an 80 mV applied potential 17. However, due to the low stability of the T7DNAP(exo-)-DNA complex under load, diminished signal * corresponding author: makeson@soe.ucsc.edu. Figure S1 (sequences of 5′-6-FAM, 3′-OH and 5′-6-FAM, 3′-H DNA oligonucleotide substrates used in gel assays); Figure S2 (unbound DNA at 70 mV applied potential); Figure S3 (primer extension gel assays supporting phi2 DNAP-DNA-dGTP ternary complex formation); Figure S4 (amplitude steps in the terminal cascade vary as a function of initial DNA substrate abasic configuration); Supporting Information Available:

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

“…The D12A/D66A mutant lacks two of the ligands for the catalytic Me 2ϩ ions in the exonuclease active site and thus has negligible exonucleolytic activity (40,41). This permits experiments to be conducted with DNA1-H_OH in the presence of Mg 2ϩ or Mn 2ϩ , conditions under which the 2Ј-H, 3Ј-OH terminated primer strand would be degraded in the bulk phase by the wild type enzyme (26,35). The extent to which p was decreased for the D12A/D66A complexes varied with the identity of the metal species; for both DNA1-H_OH and DNA1-H_H, Mn 2ϩ caused the largest decrease in p, followed by Ca 2ϩ and then Mg 2ϩ (Fig.…”

“…Nanopore Methods-Nanopore experiments were conducted as described (23,25,(35)(36)(37)(38). A single ␣-hemolysin nanopore is inserted in a ϳ25-m diameter lipid bilayer that separates two chambers (cis and trans) containing buffer solution (10 mM K-Hepes, pH 8.0, 0.3 M KCl, and 1 mM DTT).…”

“…The extent to which the abasic reporter augments the amplitude of captured complexes depends upon its position relative to the limiting aperture of the nanopore lumen. Thus, displacement of the abasic reporter group in the nanopore lumen is manifested as a change in measured ionic current (23,35).…”

“…Individual complexes reside atop the nanopore for tens of seconds, during which the measured ionic current fluctuates on the millisecond time scale between two amplitude levels (inset). Transition between the two amplitudes corresponds to movement of the DNA substrate relative to the enzyme and the nanopore by the distance of a single nucleotide (23,35,39). This spatial displacement of the DNA substrate that occurs during the translocation is detected in the time traces of ionic current by the use of a reporter group of five consecutive abasic (1Ј-H, 2Ј-H) residues in the template strand (red circles in the schematic).…”

Controlling molecular transport through nanopores

“…Combination of a-haemolysin with processive enzymes was recently demonstrated [54,55], so this approach holds great promise for DNA sequencing. It is even possible to map the extension of DNA templates with 0.5 nm resolution [56][57][58]. This is another indication of the versatility of the nanopores sensing approach and its great sensitivity for changes on molecular length scales.…”