Controlling DNA dwell time

1. 30 20 10 Translocation at constant voltage with dwell time extension with dwell time aggregation 1 10 100 1000 30 20 10 pApApA 1 10 100 1000 30 20 10 1 10 100 1000 Dwell Time (msec) Feedback Control of DNA Hairpin Molecules in a Nanopore by Elizabeth Koch Advisor: Bill Dunbar SURF-IT ‘07 Jack Baskin School of Engineering, University of California at Santa Cruz Motivation The ability to efficiently sequence DNA has huge benefits for both medicine and genomic research. While current methods require chemical processing and amplification of DNA samples, the use of nanopore devices could permit much faster and less expensive sequencing. Controlling DNA dwell time Single stranded DNA translocations occur too fast to provide enough signal to for accurate nucleotide sequencing. Dynamic voltage control implemented as a finite state machine on reprogrammable hardware allows us to control the dwell time of events: • Overview • The protein channel α-hemolysin inserted in a phospholipid bilayer allows the passage of single stranded DNA molecules driven by an electric field. • Decrease in current is seen when the pore is blocked. • The pore’s constricting size forces the molecule to travel through one nucleotide at a time. Along with corresponding changes in the current, the single-file movement is the basis for a potential DNA sequencing method [1]. This research presents the use of feedback control to detect and react to single molecule DNA in the nanopore. The opening of α-hemolysin is 2.6nm wide, allowing entrance of double stranded DNA into the vestibule Width of the hairpin prevents the whole molecule from moving through the pore until it unzips The pore constricts to 1.5nm, only large enough for ssDNA • Fishing for Klenow • DNA hairpins create a binding site for Klenow fragment, causing long dwell times before Klenow dissociates from the DNA. This produces a terminal step in the current (pictured to the right). Dynamic voltage control reacts in less than 2 msec to repeat the process rapidly using the same molecule. • Klenow fragment is a part of polymerase I, an enzyme active in DNA replication. Complimentarity of dNTPs to the template base at Klenow’s binding site cause detectable dwell time increases [2]. The interaction of these components could yield sequencing capability. Dwell time extension control is accomplished by decreasing the voltage from 180mV to 150mV when a hairpin is detected in the pore. The scatter plot in the middle shows both an increase in dwell time and a decrease in mean amplitude, which becomes dominated by lower current after the hairpin detection. The plot on the bottom shows aggregation control, in which DNA is expelled from the pore 10 msec after its detection [3]. Extended dwell times can facilitate sequencing with machine learning methods [4]. DNA event enzyme event Voltage reverses, stopping translocation Graphics thanks to Noah Wilson and Ayla Solomon References 1 Deamer, D. and Branton, D. (2002). Characterization of nucleic acids by nanopore analysis. Acc. Chem. Res., 35:817-825. 2 Benner, S., Chen, R. J., Wilson, N. A., Abu-Shumays, R., Hurt, N., Lieberman, K. R., Deamer, D. W., Dunbar, W. B., and Akeson, M. (2007). Sequence specific detection of DNA polymerase binding using a nanopore-based state machine. Submitted to Nature Nanotechnology. 3 Wilson, N. A., Abu-Shumays, R., Benner, S., Koch, E., Dunbar, W. B. (2007). Finite State Machine Control of Individual DNA Hairpin Molecules in a Nanopore. Submitted to IEEE-NIH Life Science Systems and Applications (LISSA’07) workshop. 4 Vercoutere, W., Winters-Hilt, S., Olsen, H., Deamer, D., Haussler, D., and Akeson, M. (2001). Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel. Nat Biotechnol, 19(3):248-52.