Salt Dependence of Ion Transport and DNA Translocation through Solid-State Nanopores

Smeets, Ralph M. M.; Keyser, Ulrich F.; Krapf, Diego; Wu, Mingjie; Dekker, Nynke H.; Dekker, Cees

doi:10.1021/nl052107w

Cited by 769 publications

(1,145 citation statements)

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“…Unlike solid-state pores where DNA capture increases current at less than 0.4 M KCl (ref. 32), DNA capture impedes ionic current at 0.3 M KCl for the α-haemolysin pore. DNA, KF and dNTPs were added to the cis chamber as indicated in the figures and tables.…”

Section: Nanopore Experimentsmentioning

confidence: 96%

Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

et al. 2007

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Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules 1-3 . Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates 4,5 . In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/ deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.We describe a nanopore device that monitors ionic current through a single protein pore inserted in a lipid bilayer (Fig. 1a). The limiting aperture of the pore is just sufficient to accommodate single-stranded DNA (ssDNA) 6,7 , and the adjacent pore vestibule can accommodate doublestranded (duplex) DNA (dsDNA) 7-9 . In the absence of DNA, the open channel current (I o ) through the α-haemolysin pore is 60 pA at 180 mV applied potential in 0.3 M KCl. DNA capture in the nanopore results in a decrease in the current (I). The DNA resides in the pore for a time (t D ) until it leaves, moving to the trans compartment (Fig. 1b). These two parameters, I and t D , together with current noise, are typically used to report results from nanopore experiments 6,10-19 .We used a nanopore instrument to probe the interaction of the Klenow fragment (KF) of Escherichia coli DNA polymerase I with its DNA substrate. This substrate is a duplex DNA formed by base-pairing of a short ssDNA primer with a longer template DNA. The KF catalyses DNA replication by the sequential addition of nucleotides to the primer strand, dictated by Watson-Crick complementarity to the template strand 20 . In contrast with earlier studies examining Exonuclease I/DNA complexes 4 and EcoRI/DNA complexes 5 , our nanopore Capture and translocation of a model DNA template (14 bp hairpin with a 36-nucleotide 5′ overhang and 2′-3′ dideoxycytidine terminus) resulted in a cluster of events with a median duration of 1 ms and an average blockade amplitude I = 20 pA (Fig. 2a). When the KF (2 μM) was subsequently added to the cis compartment under conditions where catalytic activity had been demonstrated in separate experiments (see Supplementary Information, Fig. S1), a second population of events emerged with a 3-ms median dwell time and a higher blockade current (I = 23 pA, Fig. 2b). This class of events is enzyme-concentration-dependent (see Supplementary Information, Fig. S2 and Table S1), consistent with nanopore capture of a DNA/ KF binary complex.Addition of a deoxynucleotide triphosphat...

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Section: Nanopore Experimentsmentioning

confidence: 96%

Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

et al. 2007

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“…The transport of small molecules and polymers across such nanopores is a very common feature in living cells and is essential to their normal function (Alberts et al 1994;Pfanner & Neupert 1990;Matouschek et al 2000;Martin et al 1991;Künkele et al 1998). Synthetic nanopores (Li et al 2003;Storm et al 2005a,b;Smeets et al 2006;Hall et al 2010;Garaj et al 2010;Schneider et al 2010) have been the focus of much interest in recent years following the demonstration of their use as effective single molecule sensors (Kasianowicz et al 1996).…”

Section: Introductionmentioning

confidence: 99%

Electro-osmotic flow through a nanopore

2014

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Electroosmotic pumping of fluid through a nanopore that traverses an insulating membrane is considered. The density of surface charge on the membrane is assumed uniform, and sufficiently low for the Poisson-Boltzmann equation to be linearized. The reciprocal theorem gives the flow rate generated by an applied weak electric field, expressed as an integral over the fluid volume. For a circular hole in a membrane of zero thickness, an analytical result is possible up to quadrature. For a membrane of arbitrary thickness, the full Poisson-Nernst-Planck-Stokes system of equations is solved numerically using a finite volume method. The numerical solution agrees with the standard analytical result for electro-osmotic flux through a long cylindrical pore when the membrane thickness is large compared to the hole diameter. When the membrane thickness is small, the flow rate agrees with that calculated using the reciprocal theorem.

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“…It senses when a single biopolymer threads it by registering a change in its ionic conductance 1,2 . The possibility of applying nanopores to the analysis of nucleic acids, in particular DNA sequencing, has generated interest 3 , and motivated fundamental studies of the physics of nanopore translocations [4][5][6][7][8][9][10][11][12][13][14][15][16][17] . The uses of solid-state nanopores have recently expanded to include detecting single proteins 18 , mapping structural features along RecA-bound DNA-protein complexes 19 and detecting spherical and icosahedral virus strains [20][21][22] .…”

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confidence: 99%

Stiff filamentous virus translocations through solid-state nanopores

et al. 2014

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The ionic conductance through a nanometer-sized pore in a membrane changes when a biopolymer slides through it, making nanopores sensitive to single molecules in solution. Their possible use for sequencing has motivated numerous studies on how DNA, a semiflexible polymer, translocates nanopores. Here we study voltage-driven dynamics of the stiff filamentous virus fd with experiments and simulations to investigate the basic physics of polymer translocations. We find that the electric field distribution aligns an approaching fd with the nanopore, promoting its capture, but it also pulls fd sideways against the membrane after failed translocation attempts until thermal fluctuations reorient the virus for translocation. fd is too stiff to translocate in folded configurations. It therefore translocates linearly, exhibiting a voltage-independent mobility and obeying first-passage-time statistics. Surprisingly, lengthwise Brownian motion only partially accounts for the translocation velocity fluctuations. We also observe a voltage-dependent contribution whose origin is only partially determined.

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Salt Dependence of Ion Transport and DNA Translocation through Solid-State Nanopores

Cited by 769 publications

References 31 publications

Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

Electro-osmotic flow through a nanopore

Stiff filamentous virus translocations through solid-state nanopores

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