SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION doi:.3/nnano..177 Replication of Individual DNA Molecules under Electronic Control Using a Protein Nanopore Felix Olasagasti, Kate R. Lieberman, Seico Benner, Gerald M. Cherf, Joseph M. Dahl, David W. Deamer and Mark Akeson* Supplementary note, Figure. Amplitude levels for the complexes mapped in panel c were determined in separate experiments that directly capture DNAP-DNA-dNTP ternary complexes formed with synthetic duplex substrates bearing 3 -H terminated primers. Because these ternary complexes are more stable than binary complexes, their duration on the pore is longer 1,3, and amplitudes can readily be determined using Clampfit software by careful inspection of the current traces. In synthesis experiments, polymerase- DNA complexes must transit through all of the stages of the catalytic cycle, including the less stable binary state. Additionally, we have observed variability in the dwell time of binary complexes at different positions along the template strands and we suspect that this effect may be dependent on the sequence context in the vicinity of the polymerase active site (data not shown). This sequence context varies as the enzyme moves along the template and we find that this effect on complex stability makes some lower amplitude events, such as the. events at position iii, too short to be detected reliably. In contrast the 7. events at position i are longer and can be assigned to an EBS during the synthesis experiments. Supplementary Movie 1. Real time current trace of T7DNAP replication of an individual DNA template deprotected upon nanopore capture. The movie shows the nanopore capture of a primer/template that was protected in bulk phase by an acridine-modified blocking oligomer. Blocking oligomer dissociation, template tethering, and controlled exposure to T7DNAP and dntp substrates were achieved as described in the text and legend for Figure 5. The first segment shows activation and replication of a single DNA template in real time (indicated as 1X in red type). Subsequent segments zoom in and slow the timescale by -fold, and then -fold (indicated as X and X, respectively), to highlight the two sequential nucleotide additions catalyzed by T7DNAP during the mv probing step on the nanopore orifice. Supplementary Movie. Real time current trace of KF replication of individually deprotected DNA templates in single-file order. The movie shows the nanopore capture of five primer/template substrates in series. Each DNA molecule was protected in bulk phase by an acridine-modified blocking oligomer. Blocking oligomer dissociation, template tethering, and controlled exposure to KF and dntp substrates were achieved as described in the text and legend for Figure. The trace shows the first seconds of the experiment in continuous real time. Individual primer/template molecules were captured for replication at 1.,., 1.9, 31., and. seconds. Supplementary Figure 1. T7DNAP replication of the DNA substrate shown in Figure a. a, Representative current trace for a captured molecule in which T7DNAP catalyzed the addition of 11 nucleotides, traversing the 1 amplitude peak in the map shown in Figure c. In the trace shown, T7DNAP rapidly stepped through multiple sequential uphill and then downhill I EBS levels while held atop the nanopore at mv applied potential. b, Fish and probe events from individual DNA molecules, showing T7DNAP replication events which progressed up and over the amplitude peak (examples 1, ), or through steps on the uphill side (examples 3, ), or the downhill side (examples 5, ), while held atop the nanopore. The event in (1) was extracted from the trace shown in panel a. Events where catalysis progressed up and over the amplitude peak (examples 1, ) were unusual. This is predicted by the relative instability of the T7DNAP-DNA complex compared to the KF-DNA complex at elevated ionic strength. 17,1 nature nanotechnology 1 Macmillan Publishers Limited. All rights reserved.

2 supplementary information doi:.3/nnano..177 Supplementary Figure. Expanded view of the current trace shown in Figure 5b. The current trace for a captured molecule in which T7DNAP catalyzed the addition of nucleotides, which is shown in a 1.5 second panel in Figure 5b, is shown here in expanded panels of.3 seconds each. Supplementary Figure 3. Expanded view of the current trace shown in Figure d. The current trace for a captured molecule in which KF catalyzed the addition of 11 nucleotides, which is shown in a 3.5 second panel in Figure d, is shown in expanded panels of.5 seconds each. nature nanotechnology Macmillan Publishers Limited. All rights reserved.