Published: January 11, 2011

r 2011 American Chemical Society 746 dx.doi.org/10.1021/nl1038874 |Nano Lett. 2011, 11, 746–750

LETTER

pubs.acs.org/NanoLett

Controlled Translocation of Individual DNA Molecules throughProtein Nanopores with Engineered Molecular BrakesMarcela Rincon-Restrepo,†,‡ Ellina Mikhailova,† Hagan Bayley,† and Giovanni Maglia*,†,§

†Department of Chemistry, University of Oxford, Oxford, OX1 3TA, United Kingdom§Department of Chemistry, University of Leuven, 3001, Heverlee, Leuven, Belgium

bS Supporting Information

ABSTRACT: Protein nanopores may provide a cheap and fast technology tosequence individualDNAmolecules.However, the electrophoretic translocationof ssDNA molecules through protein nanopores has been too rapid for baseidentification. Here, we show that the translocation of DNA molecules throughthe R-hemolysin protein nanopore can be slowed controllably by introducingpositive charges into the lumen of the pore by site directed mutagenesis.Although the residual ionic current during DNA translocation is insufficientfor direct base identification, we propose that the engineered pores might beused to slow down DNA in hybrid systems, for example, in combination withsolid-state nanopores.

KEYWORDS:Alpha-hemolysin,DNA sequencing, nanopore, protein engineer-ing, DNA translocation

The ability to sequence the human genome rapidly (e.g., in 15min) and at an affordable price (e.g., U.S. $1000) would be a

turning point in medicine. At that price and speed, most peoplecould afford to have their genome sequenced on-the-fly for tailoredmedical treatment. Thanks to advances in second-generation DNAsequencing techniques, the cost of sequencing an entire humangenome is now about U.S. $50 000.1,2 However, most second-generation technologies such as those implemented by 454Life Sciences (Roche), Solexa (Illumina) and Applied BiosystemsSOLiD (Life Technologies), rely on slow iterative cycles ofenzymatic processing and imaging-based data collection. There-fore, the most likely candidates to cross the U.S. $1000 and 15 minhuman genome barrier will be third-generation single-moleculesequencing platforms, such as Pacific Bioscience’s single molecule-real time sequencing by synthesis (SMRT) or nanopore sequencing,3

which are based on the continuous determination of sequences ofindividual DNA molecules by cycle-free processes.

In one approach to nanopore sequencing, a single-strandedDNA or RNA molecule is driven electrophoretically through ananopore and each DNA base is read as it passes a recognitionpoint.3 In its simplest manifestation, the current associated with thepassage of ions (e.g., Kþ and Cl-) through the nanopore duringDNA translocation provides the electrical read-out required todistinguish each base. In our laboratory, we use R-hemolysin(RHL) protein nanopores reconstituted in planar lipid bilayers(Figure 1). Notably, we have shown that the four DNA bases,4-6

including their epigenetically modified forms, 5-methylcytosineand 5-hydroxymethylcytosine,7 can be discriminated in a DNAstrand immobilized within the nanopore.

Alternatively, single nucleotides are identified by reading thetunnelling current passing through individual DNA bases, while aDNA strand is translocating through a solid-state nanoporemodified with tunnelling probes.8-10 Tunnelling readings wouldbe advantageous because the nanoampere range of tunnellingcurrents will allow the reading of nucleotides at a greater speedthan with the picoampere ionic currents through protein nano-pores.3 In addition, since the tip of the probe can be less than 1 nm

Figure 1. Section through a 7R7 nanopore. The amino acids at positions113, 115, 117, 119, 121, 123, and 125 were replaced in the WT7

nanopore (PDB:7AHL) by arginine by using PyMOL software (DeLanoScientific LLC, v1.0). 7R7 is in the RL2 background in which lysine 8 isreplaced by alanine as shown here. Negatively charged residues arecolored in red and positively charged residues in blue.

Received: November 4, 2010Revised: December 20, 2010

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in diameter,3 a single nucleotide would be addressed by thetunnelling probe at any given time.

One of themain remaining challenges in nanopore sequencing isto reduce the speed of DNA translocation through the nanopore,which is too fast to allow discrimination between individualnucleobases by using either ionic11 or tunnelling3 currents. Previousattempts to reduce the speed of free DNA translocation haveincluded the use of low temperatures12 and increased viscosity.13,14

These approaches reduced the speed of DNA translocation by 1order of magnitude or less, at the expense of a large decrease in theionic current. In this work, we show that by lining the transmem-brane region of the RHL pore with positively charged residues, thespeed ofDNA translocation can be slowed bymore than 2 orders ofmagnitude. Although the ionic current duringDNA translocation isagain almost completely suppressed, we suggest that such nano-pores could be used to control the speed of DNA translocation inhybrid protein and solid-state nanopores.Ionic Currents through rHL Nanopores. The heptameric

RHL nanopore contains a cap domain and a barrel domain, whichare separated by an inner constriction that is 1.4 nm in diameter(Figure 1).15 We made a variety of homo- and heteroheptamericRHL pores in which the charge distribution within the barrel wasaltered. In homoheptamers, the mutations appear in all sevensubunits, while in heteroheptamers the charge is changed in a subsetof the subunits. All mutants were made by using RL2 genes astemplates, except for theNN7 pore, in which theWTgenewas used(Supporting Information).WT pores contain an additional positivecharge at position 8 in the form of a lysine residue (Figure 1).The unitary conductance values (g) in 1 M KCl solutions

(containing 25 mM Tris-HCl and 100 μM EDTA at pH 8.0)varied greatly among the nanopores (Table 1). The introduction ofpositive charges close to the constriction increased the conductanceof the nanopores at positive applied potentials with respect to theWT7 and RL27 pores. By contrast, when positive charges wereintroduced close to the trans exit of the pore, the ionic current atpositive potentials was reduced significantly16 (Table 1). Therectification ratios (gþ/g-), calculated from the value of the ionic

current atþ50 mV divided by the current at-50 mV, also varieddepending on the position of the introduced charges. Nanoporeswith additional positive charges near the trans exit (after position121) showed gþ/g- < 1, while mutants with positive charges closeto the constriction displayed rectifying behavior similar to that ofWT7 (gþ/g- >1). On the other hand, NN7 pores, which have nocharged groups at the central constriction, displayed gþ/g-∼ 1. Inagreement with previous findings,17-19 these results suggest thatthe transport of ions through the relatively narrow RHL nanoporecan be strongly influenced by the introduction of charged residueswithin the β barrel.DNA Translocation through Homoheptameric rHL Nano-

pores. The addition of a 92mer ssDNA designed to contain nosecondary structure (50-AAAAAAAAAAAAAAAAAAAAATTC-CCCCCCCCCCCCCCCCCCCCTTAAAAAAAAAATTCC-CCCCCCCCTTAAAAAAAAAATTCCCCCCCCCC-3) toWT7

and RL27 pores from the cis chamber under positive appliedpotentials provoked current blockades that are due to the translo-cation of individual DNAmolecules through the pore20 (Figure 2a).Atþ120 mV, a most likely translocation time, tP, which is definedas the peak of a Gaussian fit to a histogram of translocation times,12

was 0.14 ( 0.01 ms for both WT7 and RL27 nanopores,16 cor-

responding to the mean most likely translocation time per base(tb = tp/number of nucleotides) of 1.5 μs/nt.The manipulation of charges in the barrel of the pore altered the

interactionbetween theDNAand the pore. The addition of one ringof arginine residues near the constriction of RL2 pores (M113R7)increased the frequency of DNA translocation by 16-fold.16 Inter-estingly, nanopores containing two or more rings of positive chargein the barrel showedonly a small additional increase in the frequencyof DNA translocation (30% for (M113R-N123)7, which was thepore that showed the highest increase, Table 1), indicating that onering of arginine residues in the barrel is sufficient to enhance thefrequency of DNA translocation to near its maximum value.16

The removal of the charges at the central constriction in NN7

or the introduction of one or two rings of arginine residues withinthe lumen of homoheptameric RL2 pores (7 or 14 additional

Table 1. DNATranslocation throughRHLNanopores atþ120mV in 1MKCl, 25mMTris-HCl containing 100μMEDTAat pH8.0a

pore tb (μs/base) f (s-1 μM-1) IRES (%) g, nS (þ120 mV)

WT7 1.5 ( 0.1 (n = 6) 3.0 ( 0.2 (n = 12) 10 (n = 5) 1.01 ( 0.01 (n = 23)

NN7 (E111N-K147N) 2.0 ( 0.1 (n = 4) 0.79 ( 0.12 (n = 4) 26 (n = 4) 1.07 ( 0.05 (n = 4)

RL27 1.6 ( 0.1 (n = 4) 0.28 ( 0.04 (n = 5) 10 (n = 4) 1.03 ( 0.02 (n = 16)

M113R7 1.5 ( 0.1 (n = 4) 4.4 ( 0.6 (n = 7) 3 (n = 5) 1.17 ( 0.02 (n = 8)

N123R7 2.4 ( 0.5 (n = 5) 0.43 ( 0.04 (n = 8) 3 (n = 4) 0.64 ( 0.02 (n = 10)

(M113R-N123)7 3.0 ( 0.3 (n = 8) 6.2 ( 1.9 (n = 7) 2 (n = 4) 0.97 ( 0.05 (n = 6)

(N123R-D127R)7 4.4 ( 0.2 (n = 5) 0.4 ( 0.05 (n = 4) 2 (n = 5) 0.57 ( 0.03 (n = 4)

(M113R-T145R)7 4.5 ( 0.7 (n = 5) 5.2 ( 1.0 (n = 5) 2 (n = 5) 1.06 ( 0.06 (n = 5)

3R7 (T115R-G119R-N123R)7 16 ( 4 (n = 7) 4.2 ( 1.4 (n = 5) <1 (n = 6) 0.87 ( 0.06 (n = 7)

4R7 (T115R-G119R-N123R-D127R)7 29 ( 6 (n = 4) 3.5 ( 3.5 (n = 4) <1 (n = 4) 0.70 ( 0.09 (n = 6)

7R7 (M113R-T115R-T117R-G119R-N121R-N123R-T125R)7 270 ( 10 (n = 6) 5.4 ( 1.6 (n = 6) <1 (n = 6) 0.91 ( 0.04 (n = 10)

7R6 RL21 120 ( 10 (n = 7) 1.54 ( 0.5 (n = 6) <1 (n = 5) 0.95 ( 0.06 (n = 7)

7R5 RL22 67 ( 10 (n = 5) 3.5 ( 0.8 (n = 4) <1 (n = 5) 0.88 ( 0.09 (n = 5)

7R4 RL23 51 ( 6 (n = 5) 3.3 ( 0.7 (n = 5) <1 (n = 5) 0.87 ( 0.05 (n = 5)

7R3 RL24 21 ( 9 (n = 7) 4.0 ( 1.5 (n = 7) 1 (n = 5) 0.94 ( 0.05 (n = 7)

7R2 RL25 9.9 ( 2.2 (n = 5) 2.3 ( 0.5 (n = 4) 2 (n = 5) 0.93 ( 0.07 (n = 5)

7R1 RL26 6.0 ( 1.1 (n = 5) 1.7 ( 0.3 (n = 5) 4 (n = 4) 0.99 ( 0.05 (n = 5)a tb is the most likely DNA translocation time per base; f is the normalized frequency of DNA translocation; g is the unitary conductance and IRES is theresidual current during DNA translocation. For IRES, the errors were all less than(1%. All mutants, except NN7, are in the RL2 background (WT porescontain an additional positive charge at position 8 in the form of a lysine residue, Supporting Information). Errors are shown as standard deviations.

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positive charges) had only a small effect on the most likelytranslocation time (tP) for the 92mer or other short ssDNA orRNA molecules.19,21 For all double arginine mutants tested(Table 1), tP was just 2- or 3-fold higher than the value observedwith WT7 and RL27 pores (Table 1). However, the introductionof three (3R7) or four (4R7) rings of arginine residues within thebarrel increased the most likely translocation time by ∼10- and∼20-fold, respectively, while seven rings of arginines residues(7R7) increased the translocation time by more than 2 orders ofmagnitude relative to the WT7 and RL27 values (Table 1). In allthe nanopores studied, the translocation times of the currentblockades induced by DNA showed a Gaussian distributionfollowed by an exponential tail (Figure 2b). The most likelytranslocation time per base decreased exponentially with theapplied potential (Figure 2c), confirming that, as for WT7 pores,individual DNA molecules are translocated through the porerather than binding and dissociating from the cis side.22-25

Residual Currents through Homoheptameric rHL Nano-pores. When a DNA molecule is translocating through a nano-pore, the ionic current is reduced from the open pore current (IO)to the blocked pore current (IB). In WT7 and RL27 nanopores theresidual current (IRES), defined as IB divided by IO expressed inpercent, is ∼10%16,20,26 (Table 1). Modification of the chargedistribution within the barrel of the pore has a powerful effect onIRES. For example, the NN7 pore in which the charged residues ofthe central constriction are substituted by smaller, neutral residuesshows an increased residual current, IRES = 26% (Table 1, Figure 3),while the introduction of large, positively charged arginine residuesgreatly reduces IRES (Table 1, Figure 3). One ring of arginine

reduces IRES to 3%, while three or more sets of arginine residuesalmost completely suppress the residual current (IRES < 1%).The effect of arginine substitutions on the residual current

during DNA translocation is most likely due to the reducedcross-section of the lumen of the pore, given the increased bulkof the arginine side chains by comparison with the side-chains inWT7 nanopores,

5 which limit the current flow of hydrated ionsthrough the pore. An additional reduction of the ionic flow is mostlikely due to the increased electrostatic self-energy of the translo-cating positive ions (e.g., Kþ)27,28 that follows the introduction ofthe positive charges in the lumen of the pore. Finally, it is alsopossible that the interaction of the positive charges of the arginineside chains with the backbone phosphates of DNA29 triggers apartial collapse of the β barrel around the DNA, which in turnprevents the passage of ions through the pore.DNA Translocation through rHL Heteroheptameric Pores.

Heteroheptameric RHL pores were prepared by mixing 7R-monomers containing an eight-aspartate tail (D8) at the Cterminus with RL2-monomers, followed by separation by SDS-PAGE (Supporting Information). When incorporated into hepta-mers, each 7R monomer provides seven arginine residues withinthe barrel (starting from the constriction and ending at the trans

Figure 2. DNA translocation through 7R7 nanopores. (a) Single-channelrecordings of RL27 (top) and 7R7 nanopores (bottom) atþ120 mV afterthe addition of 1.0 μM 92-mer ssDNA to the cis compartment. (b) Eventhistogram showing the translocation time distribution at þ120 mVthrough a 7R7 pore upon addition of 0.7 μM ssDNA (92-mer) to thecis compartment. Events of <10 ms are attributed to the transient gating ofthe 7R7 nanopore and are ignored (Supporting Information Figure S1).The solid line shows a fit to the histogramof aGaussian followedby a singleexponential.12 The most likely translocation time (tP) is defined by thepeak of the Gaussian fit.12 (c) Dependence on the applied voltage of themost likely translocation time per base (tb = tP/92) of ssDNA (92 mer)through 7R7 nanopores. The red line shows a single exponential fit.Experiments were performed in 1 M KCl, 25 mM Tris-HCl, containing100 μM EDTA, at pH 8.0.

Figure 3. Residual current (IRES) throughRHLnanopores during ssDNAtranslocation atþ120 mV. Errors are expressed as standard deviations. Allnanopores, except WT7 and NN7, are in the RL2 background. Nanoporeswith additional positive charge compared to theWT7 andRL27 pores are inblue. Experiments were performed in 1 M KCl, 25 mM Tris-HCl,containing 100 μM EDTA, at pH 8.0.

Figure 4. Dependence of themeanmost likely translocation time per base(tb) atþ120 mV on the number of arginine residues in the barrel of RL2pores. Homo- and heteroheptamers are shown in open circles and bluetriangles, respectively. tb is expressed on a logarithmic scale and the data arefitted to a linear regression. Heteroheptamers were obtained by mixingRL2- and 7R- monomers. Experiments were performed in 1 M KCl, 25mM Tris-HCl, containing 100 μM EDTA, at pH 8.

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exit of the pore). It was hoped that the arginine side chains wouldinteract with the phosphodiester linkages of DNA and reduce therate of translocation, while the relatively small side-chains of theRL2-subunits would still allow the passage of a significant ioniccurrent.We found that heteroheptameric pores containing 7R mono-

mers were as efficient as homopolymeric pores containing arginineresidues at reducing the rate of translocation of DNA throughthe pore (Figure 4 and Table 1) with the translocation timesper base showing an exponential dependence on the number ofarginine residues in the barrel (Figure 4). Unfortunately, IRES wasagain reduced to almost zero when 21 or more arginine residueswere introduced into the barrel of the pore (Table 1).Conclusions. In this work, we have shown that molecular

brakes in the form of additional positive charge can be engineeredwithin the barrel of a protein nanopore to control the average speedof translocation of a short ssDNA molecule containing no second-ary structure. Although the introduction of more than two posi-tively charged residues per subunit in homoheptamers or morethan two 7R subunits in heteroheptamers largely prevented thepassage of ions through the pore, the results obtained here may stillbe useful for the implementation of nanopore sequencing plat-forms. For example, an artificial barrel containing positive chargescould be paired with a pore capable of base recognition (e.g.,NN7).

4 The charge within the artificial barrel would be used tocontrol DNA translocation, while the trans-membrane pore wouldrecognize single DNA bases (Figure 5a). In such a construct, webelieve that substantial current will flow, as previous studies haveshown that proteins handlingDNAplaced at the cis entrance to theRHL nanopore do not drastically alter the ionic current throughthe pore and allow bases to be discriminated4,30-32

Alternatively, modified RHL pores could be inserted into solid-state nanopores33 paired with suitable tunnelling probes34-37

(Figure 5b). The feasibility of this approach has been describedtheoretically38-42 and the ability of scanning tunnellingmicroscopyto recognize single bases has been shown experimentally.43-45

However, to be identified during translocation through a nanopore,each DNA base would have to be addressed by a tunnelling probefor at least 100 μs,3 which is 3 orders of magnitude longer that thetypical average translocation times of individual DNA bases or basepairs through solid state nanopores.3,46-48 In addition, a reprodu-

cible orientation and positioning of the base at the tunneling probewill also be crucial, as electron-tunneling currents are exponentiallysensitive to atomic scale changes of orientation and distance.3

Therefore, if tunnelling probes can be incorporated into solid-statenanopores with atomic precision, the system shown in Figure 5bwill be capable of informative data acquisition (tb = 270 μs/base atþ120mV), providing that the speed ofDNA translocation throughthe pore is constant and the orientation of each base at thetunnelling probe reproducible.3

’ASSOCIATED CONTENT

bS Supporting Information. Additional results, methods,and a figure are included. This material is available free of chargevia the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*E-mail: giovanni.maglia@chem.kuleuven.be.

Present Addresses‡Institute of Bioengineering and Swiss Institute of ExperimentalCancer Research, Ecole Polytechnique F�ed�erale de Lausanne,Lausanne, Switzerland.

’ACKNOWLEDGMENT

We are grateful to David Stoddart and to Gabriel Villar forproof reading and insightful comments and for helping with datafitting. This work was funded by the National Institutes ofHealth.

’REFERENCES

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Figure 5. Potential uses of 7R7 nanopores. (a) A protein nanopore (e.g.,NN7, green) is used to detect base-specific ionic current differences in aDNA strand, while a synthetic protein barrel (red) containing positivecharges is employed to control the speed of DNA translocation. (b) Aprotein nanopore with positive internal charge (e.g., 7R7, red) is pairedwith a solid-state nanopore (gray) equipped with a tunnelling probeintegrated into the device with atomic precision. The speed at which DNAis translocated through the solid-state nanopore is controlled by the appliedpotential and the number of positive charges in the barrel of the proteinnanopore. Each base will be read by monitoring the tunnelling current,providing that the bases translocate at a constant speed and arrive with thesame orientation at the tunnelling probe.

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