Controlling DNA Capture and Propagation through Artificial Nanopores Eliane H. Trepagnier, ²,‡,§, | Aleksandra Radenovic, ²,§, | David Sivak, ‡,⊥ Phillip Geissler, ‡,⊥ and Jan Liphardt* ,‡,§,| Biophysics Graduate Group, UniVersity of California, Berkeley, Berkeley, California, Department of Physics, UniVersity of California, Berkeley, Berkeley, California, Physical Biosciences DiVision, Lawrence Berkeley National Laboratory (LBL), Berkeley, California, and College of Chemistry, UniVersity of California, Berkeley, Berkeley, California Received June 15, 2007; Revised Manuscript Received July 30, 2007 ABSTRACT Electrophorescing biopolymers across nanopores modulates the ionic current through the pore, revealing the polymer’s diameter, length, and conformation. The rapidity of polymer translocation (∼30 000 bp/ms) in this geometry greatly limits the information that can be obtained for each base. Here we show that the translocation speed of λ-DNA through artificial nanopores can be reduced using optical tweezers. DNAs coupled to optically trapped beads were presented to nanopores. DNAs initially placed up to several micrometers from the pore could be captured. Subsequently, the optical tweezers reduced translocation speeds to 150 bp/ms, about 200-fold slower than free DNA. Moreover, the optical tweezers allowed us to “floss” single polymers back and forth through the pore. The combination of controlled sample presentation, greatly slowed translocation speeds, and repeated electrophoresis of single DNAs removes several barriers to using artificial nanopores for sequencing, haplotyping, and characterization of protein-DNA interactions. The characterization of single biopolymers is one of the potential applications of artificial nanopores 1,2 and nano- channels. 3-5 For example, artificial nanopores have been proposed as tools for rapid DNA sequencing. 1,2,6-10 This approach is based on the modulation of the ionic current through the pore as a polymer traverses it, revealing the polymer’s diameter, length, and conformation. 1,2,6-10 A road- block to single base resolution in such nanopore-based DNA sequencing approaches is a lack of control over translocation speeds. Speeds can be reduced by lowering the ionic strength or the driving voltage, 11 but this comes at the cost of decreased ionic current, reducing the signal-to-noise ratio. The widespread use of optical tweezers in single-molecule biophysics suggests their utility to control sample presenta- tion to a pore or channel, reduce polymer propagation speeds without impairing ionic currents, and to repeatedly character- ize one DNA molecule. The integration of optical trapping with synthetic nanopore translocation experiments was first demonstrated by Keyser et al. 12 These authors used optical force measurement to calculate the electrical force exerted on a DNA molecule in a nanopore as well as the effective charge of a DNA molecule in a nanopore. Here we extend their results by showing that this geometry can be used to reduce translocation rates by several hundred-fold and also repeatedly translocate and retract a single DNA. Optical tweezers are routinely used to study mechanical properties of biopolymers (for a review, see ref 13; the details of our optical tweezers instrument are described in ref 14 and in the Supporting Information). Typically, a molecule is connected by complementary chemistry to a micrometer-scale bead that can be manipulated in three dimensions by one or more highly focused laser beams. In our experiment, λ-DNAs were connected to 10 μm polysty- rene beads via streptavidin-biotin linkage. As shown in the chamber schematic (Figure 1A), the trapped beads were brought into proximity of a single artificial nanopore in a membrane separating two chambers. Electrodes maintained an electrical potential across the nanopore. Nanopores were coated via atomic layer deposition (ALD) with 2-15 nm of alumina, 15 a thermally and chemically stable insulating dielectric material. At our pH values (pH ) 7.0-8.0) and * To whom correspondence should be addressed. E-mail: Liphardt@ physics.berkeley.edu. ² These authors contributed equally to this work. ‡ Biophysics Graduate Group, University of California, Berkeley. § Department of Physics, University of California, Berkeley. | Physical Biosciences Division, LBL. ⊥ College of Chemistry, University of California, Berkeley. NANO LETTERS 2007 Vol. 7, No. 9 2824-2830 10.1021/nl0714334 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/18/2007