Reduction of DNA Folding by Ionic Liquids and Its Effects ...

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Reduction of DNA Folding by Ionic Liquids and Its Effects on the Analysis of DNA–Protein Interaction Using Solid-State Nanopore

Ki-Baek Jeong, Ke Luo, Min-Cheol Lim, Jong-Yoon Jung, Jae-Seok Yu, Ki-Bum Kim, and Young-Rok Kim*

Dr. M.-C. LimFood Safety Research GroupKorea Food Research InstituteSungnam 13539, South KoreaJ.-S. Yu, Prof. K.-B. KimDepartment of Materials Science and EngineeringSeoul National UniversitySeoul 08826, Korea

DOI: 10.1002/smll.201801375

Nanopore sensing platform has also been utilized for investigating the complex-ation between biomolecules of interest in aqueous environment in search of specific biomarkers.[10] The types of complexation include DNA–DNA,[11] RNA–proteins,[12] and DNA–proteins. In particular, RecA,[13] histone,[14] zinc finger protein (ZFP),[15] α-thrombin,[16] antibodies,[10] and methyl-binding protein (MBP)[17] are among the proteins that form a specific interaction with DNA, and their specific affinities as well as the loci of binding are of interest in biological and biomedical area.

However, there are several technical challenges that need to be resolved to achieve high-resolution detection of target biomolecules in nanopore analysis. DNA is a long polymeric molecule that is often present in entangled form in aqueous solution of which the degree of entan-glement is proportional to the length of DNA.[18] The extremely high voltage bias

applied to the vicinity of nanopore could also cause folding of DNA during translocation even though the diameter of nanopore is much smaller than the persistence length of DNA (≈50 nm).[19,20] DNA folding is a pheno menon in which a strand of DNA is forced to collapse during the translocation through a nanopore with a diameter smaller than the persis-tence length of DNA by strong electrical dragging force. Such an entanglement or folding of DNA interferes with the precise analysis of the complexation of DNA with a specific protein or complementary nucleic acid because the additional current blockade that reflects the presence or location of bound bio-molecules along the strand of DNA is buried by the large cur-rent blockades derived from the DNA folding taking place near

DNA folding is not desirable for solid-state nanopore techniques when analyzing the interaction of a biomolecule with its specific binding sites on DNA since the signal derived from the binding site could be buried by a large signal from the folding of DNA nearby. To resolve the problems associated with DNA folding, ionic liquids (ILs), which are known to interact with DNA through charge–charge and hydrophobic interactions are employed. 1-n-butyl-3-methylimidazolium chloride (C4mim) is found to be the most effective in lowering the incident of DNA folding during its translocation through solid-state nanopores (4–5 nm diameter). The rate of folding signals from the translocation of DNA–C4mim is decreased by half in comparison to that from the control bare DNA. The conformational changes of DNA upon complexation with C4mim are further examined using atomic force microscopy, showing that the entanglement of DNA which is common in bare DNA is not observed when treated with C4mim. The stretching effect of C4mim on DNA strands improves the detection accuracy of nanopore for identifying the location of zinc finger protein bound to its specific binding site in DNA by lowering the incident of DNA folding.

Nanopore Sensing

K.-B. Jeong, K. Luo, J.-Y. Jung, Prof. Y.-R. KimInstitute of Life Sciences and ResourcesDepartment of Food Science and BiotechnologyKyung Hee UniversityYongin 17104, South KoreaE-mail: [email protected]

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smll.201801375.

1. Introduction

Solid-state nanopore is an ultrasensitive sensing platform that allows a single molecule detection with high throughput by probing the structural information of the target mole cules while they are electrophoretically driven through a nanometer-sized pore.[1,2] During the translocation of biomolecules through a nanopore, transient alterations of ionic current occurs, which reflect the physical information of the analytes.[3,4] With the sta-tistical analysis of the ionic current traces, the solid-state nano-pore has been widely used in the characterization of various analytes such as DNA,[5,6] RNA,[7] and proteins[8,9] at single molecule level.

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to their binding sites. Therefore, reducing the occurrence of folding event during DNA translocation would enhance the accuracy of signal readouts associated with the bound biomolecules on DNA strand. It has been reported that the persistence length of DNA is influenced by the interactions between DNA bases,[21] which would also affect the rate of folding phenomenon of DNA. Here, we employed three ionic liquids (ILs), 1-ethyl-3-methylimidazolium chloride [(C2mim)Cl], 1-butyl-3-methylimidazolium chloride [(C4mim)Cl], and 1-octyl-3- methylimidazolium chloride [(C8mim)Cl], which have been reported to bind to the minor groove of double helical structure of DNA to increase the persistence length of DNA, and thus lower the occurrence of DNA folding during translocation.

IL, a salt in liquid state, is composed mainly of bulky and asymmetric organic cations paired with a weakly coordinating anion. It has been reported to be an effective reagent for the extraction, separation, and preservation of DNA due to its intrinsic interactions with DNA, which are derived from the electrostatic interaction between ILs and DNA.[22,23] The hydro-phobic interaction between alkyl chains of ILs and the bases of DNA also play an important role in formation of DNA–ILs complexes.[24,25] In this study, we employed ILs paired with chloride ion to minimize the effect of anions on the structural conformation of DNA. The anions of ILs have been reported to interact with the bases of DNA through hydrogen bond, of which the degree of interaction is determined by the kosmo-tropic and chaotropic nature of the anion species.[26,27] But, chloride ion represents the border line case between salting-in and salting-out species according to the Hofmeister series.[28–30]

Recently, it has been reported that the translocation speed of DNA through nanopore could be reduced in the presence of ILs.[31,32] The positive charge of immidazolium group in IL is believed to counterbalance the negative charges on the phos-phate groups in DNA backbone, resulting in the reduction of

overall negativity of DNA and thus slowing down the transloca-tion speed through nanopore at a given voltage bias.

In this study, the effects of ILs with different alkyl chain length on the conformation of DNA and their mechanisms were inves-tigated using solid-state nanopore. The conformational changes of DNA through complexation with ILs were further examined by atomic force microscopy (AFM) and fluorescence microscopy analysis. ILs were shown to be effectively lowering the occurrence of DNA folding during translocation events, thereby improving the resolution of electrical readout for identifying the specific binding of zinc finger protein on DNA with its binding sites.

2. Results and Discussion

The translocation behavior of DNA with and without ILs through nanopore was investigated using the nanopore fabricated on 10 nm thick silicon nitride (SiN) membrane. A nanopore (4–5 nm diameter) was drilled onto the SiN membrane by a highly focused electron beam using transmission electron microscope (TEM) according to the methods reported in previous study.[15] The nano-pore was placed in between two chambers filled with 1 m KCl-TE (pH 8.0) where DNA samples were introduced to cis chamber (Figure 1a). The DNA and DNA–ILs were electrophoretically driven through the nanopore under the applied voltage of 150 mV across the nanopore membrane. Translocation of DNA through the nanopore was monitored as a transient blockade of ionic current. These events were interpreted in terms of the depth of conductance blockade, ΔG, and its dwell time, Δτ.

Figure 1b shows typical conductance blockade events for bare DNA (991 bp) and C4mim-treated DNA (DNA–C4mim) under a bias voltage of 150 mV. The current drop histograms were fitted by a Gaussian function with a mean current drop of 2.22 and 5.25 nS for type I and type II event, respectively

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Figure 1. Nanopore analysis of DNA (991 bp) with 100 × 10−3 m of C4mim through 5 nm pore at bias voltage of 150 mV in 1 m KCl. a) Experimental setup illustrating the translocation of DNA from cis to trans chamber through nanopore under voltage bias across the membrane. Inset shows TEM image of the nanopore (4.8 nm diameter) drilled by highly focused electron beam. The linear I–V curves, indicating the ohmic nature of the nanopore are shown below. The diameter of two nanopores used for the analysis of bare DNA and DNA–C4mim was almost same as indicated by the overlapping I–V curves. b) Current traces from the translocation of bare DNA and DNA–C4mim. The level of current blockades classified as type I and type II are indicated in red and blue dotted line, respectively. c) Histogram of conductance blockades from the translocation of bare DNA and DNA–C4mim. The type I (red) events represent the translocation of DNA in stretched form whereas type II (blue) events represent that in folded form.

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(Figure 1c). The mean current blockade of type II events was nearly twofold higher than that of type I events, suggesting that type II signals were derived from the translocation of DNA in folded form.[20,33] Based on the statistical analysis, the propor-tion of events derived from the translocation of bare DNA in folded form (type II) was calculated to be ≈31% from 644 trans-location events total. However, the proportion of type II events in DNA–C4mim was significantly decreased down to 15%. The results suggest that the DNAs complexed with ILs, C4mim, are less likely to form folded structure during nanopore anal-ysis possibly due to the increased persistence length of DNA. The binding of ILs to the minor groove of double-stranded DNA would transform the DNA into more rigid state.[25,34] The possible conformation of DNA bound with C4mim is shown in Figure 2a. In molecular dynamic (MD) simulation, the bases in DNA strands were reported to be disturbed by the binding of ILs to the minor groove of DNA while the helical structure of double-stranded DNA is well maintained.[25] In addition, the binding Gibbs energy of ILs to DNA is influenced by the alkyl chain length of the ILs.[23] The binding of ILs to the double-stranded DNA was further evidenced by the fact that DNA–C4mim complex was resistant to the enzymatic hydrolysis by DNase (Figure S1, Supporting Information). Bare DNAs were readily hydrolyzed by DNase whereas the DNAs com-plexed with ILs were not affected by the same concentration of DNase, implying that the binding or catalytic activity of DNase was inhibited by the ILs that preoccupied the binding site, grooves of double-stranded DNA. On the other hand, the DNase hydrolyzed the DNA when the same ILs were added simulta-neously with DNase right after mixing each other, suggesting that ionic liquid itself does not inhibit the catalytic activity of

the DNase but blocks the binding of the enzyme which is a pre-requisite for the enzymatic hydrolysis. It is very likely that the binding of DNase and enzymatic hydrolysis of DNA was taking place before ILs took a position in the grooves of dsDNA when ILs and DNase were introduced simultaneously.

The translocation speed of DNA was also influenced by ILs. As shown in Figure 2b, the average dwell time of DNA–C4 mim (τd = 0.332 ms) was approximately two times longer than that of bare DNA (τd = 0.178 ms). The scatter plots displaying indi-vidual translocation events also showed that the proportion of DNA translocation in folded form was higher in bare DNA in comparison to DNA–ILs (Figure 2c). The ILs complexed with DNA would increase the hydrodynamic radius of DNA since the molecular size of C4mim+ (length, 11.0 Å; width, 5.8 Å)[35] is much larger than that of K+ ion (1.33 Å) or water.[36] The positive charge of ILs that counterbalance the negative charge of phosphate groups in DNA backbone would also be respon-sible for the decreased dwell time of DNA.[32] While C4mim effectively lowered the incident of DNA folding during nano-pore analysis, the other ILs, C2mim and C8mim, exhibited distinctively different results. No translocation events were observed when DNA was complexed with C2mim. For C8mim, the number of translocation events was also significantly lower than the bare DNA or DNA–C4mim. It is also interesting to note that the translocation events with several-fold higher conductance blockade signal was dominant when DNA was complexed with C8mim in comparison to the one obtained from bare DNA (Figure S2, Supporting Information).

To investigate the role of alkyl chain length in ILs on struc-tural conformation of DNA, three different ILs were selected and their effects on the structure of model DNA, λ-DNA (48.5 kbp),

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Figure 2. Statistical analysis of the events from the translocation of bare DNA and DNA–C4mim. a) Schematics illustrating the possible conforma-tion of DNA with and without C4mim. The DNA with C4mim is less likely to form folded structure during translocation. b) Histogram of dwell time in log scale for bare DNA and DNA–C4mim. c) Scatter plot displaying individual translocation events of bare DNA (red) and DNA–C4mim (blue) (both 991 bp). The distribution of events reflecting the DNA translocation in folded and stretched form were clustered by ellipses. Of all translocation events, the percentage of events in folded form is shown.

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were examined using AFM and fluorescence microscopy. Figure 3a shows AFM and fluorescence microscopy images of DNAs treated with three different ILs, C2mim, C4mim, and C8mim. DNA without ILs was also examined as a control. For AFM analysis, all the DNA samples with and without ILs were applied onto mica surface. DNA with ILs showed dis-tinctive conformational changes in comparison to the control bare DNA. For bare DNA, individual DNA strands were ran-domly spread over the mica surfaces with a frequent tangled knot structures that might be induced by coiled aggregation of DNA strands. The DNA treated with C2mim was shown as a highly aggregated form and no individual DNA strands were observed. It clearly explains the absence of translocation events when the DNA was treated with C2mim. The conformation of DNAs treated with C4mim was in part similar to bare DNA but the occurrence of DNA tangle was dramatically reduced. On the other hand, it is interesting to note that the DNAs treated with C8mim showed bundle structures, suggesting that there is a favorable interaction between individual DNA strands. The formation of bundle structure was more evident in fluo-rescence microscopy image, showing that long λ-DNAs were aligned together in stretched bundle form in aqueous solution. It is speculated that the cationic immidazolium head group of C8mim is in contact with the negatively charged phosphate backbone of DNA whereas the end part of long alkyl chain in C8mim are facing away from the DNA strand, inducing hydro-phobic interactions with the ILs’ alkyl chains attached to the neighboring DNA strands. In case of C4mim, the alkyl chain is

not long enough to form massive hydrophobic interactions with those attached to the neighboring DNA. The electrostatic inter-action between cationic immidazolium group of C4mim and negatively charged phosphate backbone of DNA accompanied by subsequent hydrophobic interaction between alkyl tail group of the IL and bases of DNA would bring about stable binding of the IL in double-stranded DNA.[37] The binding of C4mim to the minor groove of double-stranded DNA would increase the persistence length of the double-stranded DNA, lowering the occurrence of DNA entanglement which is quite evident in AFM image. However, the DNA treated with C2mim showed quite different conformation compared with that treated with C4mim. We assumed that the difference would be originated from the net charge of DNAs after complexing with each ILs thus we performed zeta potential analysis to validate our hypothesis. As expected, the zeta potential of DNA decreased from −40 mV to close to neutral after complexing with C2mim at test concentration (100 × 10−3 m) (Figure S3, Supporting Information). The net charge of DNA remained constant at around neutral range at the IL’s concentration over 100 × 10−3 m. On the other hand, the zeta potential of C4mim–DNA is also lower than that of bare DNA but the degree of reduction is not as significant as that treated with C2mim. The DNA treated with the same concentration of C4mim still presented net negative charge of around −20 mV. The electrical neutraliza-tion of DNA after complexing with C2mim is believed to be responsible for the aggregation of DNA. That is, the number of C2mim bound to a given length of DNA is much greater than

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Figure 3. Conformational change of DNA upon complexation with ILs. a) AFM and fluorescence microscopy images of bare DNA, DNA–C2mim, DNA–C4mim, and DNA–C8mim. λ-DNA was used in this analysis. The field sizes of AFM images are 3 × 3 µm with Z-scale of 6 nm. b) Schematics illustrating the possible conformation of DNA complexed with ILs of different tail length; C2mim, C4mim, and C8mim.

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C4mim probably due to the smaller molecular size of C2mim, which could effectively cancel off the negative charge of DNA. The steric hindrance taking place between C4mims bound on DNA would limit the number of ILs that could complex with DNA, resulting in the saturation of charge reduction at the concentration over 100 × 10−3 m. It has been reported that the accumulation behavior of the anion species around biomol-ecules differs depending on the sizes of anions in the ILs.[27] That is, large coordinating anions like acetate brought about a strong denaturation effect on biomolecules whereas smaller chloride ion induced minor distortion due to the small size and weaker interactions with the biomolecules. In this study, we employed ILs coordinated with chloride ion to minimize the effect of anions on the structure of DNA. It is also worth noting that chloride ion generally represents a border line case between salting-out and salting-in effect on biomolecules according to Hofmeister series.[28–30] In addition, the concentra-tion of chloride ions derived from tested ILs is 100 × 10−3 m, which is trivial considering that the concentration of chloride ions in base electrolyte (1 m KCl) is tenfold higher. Thus, it is very likely that the changes in the conformation of DNA are mainly caused by the interaction of cationic ILs with DNA.

We further investigated the effect of ILs on improving the resolution of signal readout when detecting the binding of ZFP to a target DNA harboring two specific binding sites for the ZFP. The amplitude of current blockade signal represents the hydrodynamic diameter of translocating polymer. When a protein is bound to a strand of DNA, additional current drop is observed, of which the position of additional current drop reflects the binding site of protein. DNA–ZFP samples were prepared by mixing target DNA (1152 bp with two ZFP-binding sites) and ZFP (zif268 NRE) to a molar ratio of 1:150 and incu-bated at 25 °C for 60 min. Then, 100 × 10−3 m of C4mim was added to the sample and incubated further for 60 min before nanopore analysis. As expected, the occurrence of folding signal was notably reduced from 32 to 21% by treating DNA–ZFP with C4mim (Figure 4). As a result of reducing the incident of DNA folding, the ratio of additional current drop signals derived from the ZFP binding to the DNA was increased from 19 to 32%. It implies that a significant number of events representing the binding of ZFP on the DNA was buried by the large current blockade derived from DNA folding. The stretching of DNA by C4mim also extended an average dwell time for the transloca-tion of DNA from ≈1.196 to ≈2.493 ms. Even though the DNA

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Figure 4. Analysis of the DNA–ZFP using a 5 nm nanopore at 150 mV in 1 m KCl. The DNA is 1152 bp long harboring two specific binding sites for ZFP. a) Current traces reflecting the translocation events of DNA–ZFP (upper) and DNA–ZFP–C4mim. Red line indicates the typical level of current blockade signals from the translocation of DNA in stretched form, whereas green line represents the level of signals induced by the protein bound on the strand of DNA. Blue line indicates the translocation signals of DNA in folded form, which is twofold higher than the one from the DNA in stretched form. Inset shows a representative current traces derived from the translocation of DNA–ZFP in folded (upper) and stretched (lower) form. b) Scatter plot of the dwell time versus conductance blockade (ΔG) for DNA–ZFP (left) and DNA–ZFP–C4mim (right). c) Histogram of dwell time for the same samples. d) Histogram of the conductance blockade (ΔG) of DNA–ZFP (top) and DNA–ZFP–C4mim (bottom). Red, green, and blue lines represent the signals originated from the translocation of stretched DNA, protein bound DNA, and folded DNA, respectively. A total 1397 and 1340 signals from the translocation of DNA–ZFP and DNA–ZFP–C4mim, respectively, were analyzed.

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folding was not completely inhibited by C4mim, the resolution of signal in nanopore analysis was notably improved. On the other hand, the ZFP bound to the specific site on DNA should not be dissociated by treating the sample with ILs. Otherwise, ILs would not be suitable for monitoring the binding of protein to a specific site on DNA. Electrophoretic mobility shift assay (EMSA) measures the mobility changes of DNA upon complex-ation with a protein. The mobility of DNA complexed with ZFP should be much lower than that of naked DNA. The mobility shift was in proportion to the number of protein bound to the DNA. With a molar ratio of 1:100 for DNA and ZFP, all the two binding sites in DNA were occupied by ZFP, while a majority of the same DNA had only one ZFP bound when the molar ration of DNA and ZFP was 1:50, which is reflected by the mobility shift in gel electrophoresis (Figure S4, Supporting Information). It implies that all the binding sites in test DNA were occupied by the ZFP at the molar ratio of over 1:100 for DNA and ZFP. The ZFPs bound onto the DNA with two spe-cific binding sites were not affected by the treatment with ILs. The position of band representing the DNA with two ZFPs was exactly same for both samples treated with and without ILs, implying that the bound ZFP on DNA is insensitive to the ILs (Figure S5, Supporting Information). If DNA–ZFP complex was destabilized or disrupted by C4mim, the mobility shift would have occurred for the sample treated with ILs. From the results, it is clear that the ILs, C4mim, do not affect the DNA–ZFP complex but improve the accuracy of signal readout for detecting the ZFP binding on DNA through inhibiting the folding of DNA during nanopore analysis.

3. Conclusion

The electrostatic and hydrophobic interaction between ILs and dsDNA led to the conformational changes in DNA, altering the translocation behavior of DNA during nanopore analysis. The length of hydrophobic tail in ILs was found to be an important factor determining the conformation of DNA upon complexa-tion. C4mim was the most effective in lowering the incident of DNA folding in aqueous solution possibly by increasing the persistence length of double-stranded DNA. The rate of DNA folding during the translocation through nanopore was decreased by half in the presence of C4mim compared with that of naked DNA. The other two ILs, C2mim and C8mim, showed distinctively different effect on the conformation of DNA. The DNAs treated with C2mim underwent severe aggregation which is not suitable for nanopore analysis. The aggregation of DNA in the presence of C2mim is believed to be a result of the charge neutralization of DNA upon complexation with a large number of C2mim, which removed the repulsive force between negatively charged DNAs. On the other hand, C8mim induced the formation of DNA bundles which is also not desirable for nanopore analysis. The long hydrocarbon tail of C8mim sticking out from the DNA–C8mim complex would be respon-sible for the intermolecular interaction, leading to the forma-tion of bundle structure. The effect of C4mim on stretching the strand of DNA improved the resolution of signal derived from the translocation of DNA–ZFP complex because the additional current blockade originated from the bound ZFP on DNA is

less likely buried by a large current blockade signal derived from the folded region of DNA. Even though the DNA folding is not completely inhibited by the IL, we believe that this is a meaningful step toward understanding their interactional prop-erties and resolving the problems associated with DNA analysis using solid-state nanopore, which would have an immense applications in the field of biological and analytical sciences.

4. Experimental SectionChemicals: Three types of ionic liquids, (C2mim)Cl, (C4mim)Cl,

and (C8mim)Cl, were purchased from Sigma-Aldrich (St. Louis, MO). Potassium chloride (KCl), ethylenediaminetetraacetic acid (EDTA), Tris(hydroxymethyl)aminoethane hydrochloride (Tris-HCl), nickel chloride (NiCl2), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), maltose monohydrate, zinc sulfate (ZnSO4), magnesium chloride (MgCl2), ethidium bromide (EtBr), and Coomassie blue were purchased from Sigma-Aldrich (St. Louis, MO). Polydimethylsiloxane (PDMS) monomer and the curing agent were purchased from Dow Corning (Midland, MI). Distilled water was used in all experiments. Agar powder and sodium chloride (NaCl) were provided from Daejung Chemicals & Metals Co., Ltd. (Gyonggi-do, Korea). Luria-Bertani (LB) broth was purchased from BD (Difco, Lawrence, KS). Ampicillin, isopropyl-β-d-thiogalactopyranoside (IPTG), and dl-dithiothreitol (DTT) were purchased from Biosesang (Gyonggi-do, Korea). The DNA ladder (100 bp) and protein ladder (10 kDa) were from Noble bio (Gyonggi-do, Korea). DNase I was purchased from GE Healthcare (Seoul, Korea).

Preparation of ZFP: The ZFP was prepared as described elsewhere.[15] Briefly, the gene encoding zif268//NRE was cloned into a pMal-c2x vector harboring a MBP tag. The plasmid vector containing ZFP was introduced into the host strain, E. coli BL21 (DE3), for overexpression of the protein. The host strain harboring the expression vector was grown in LB with ampicillin (100 µg mL−1) at 37 °C with shaking (180 rpm).[15] Upon reaching to an OD600 of 0.7, the culture was induced with IPTG to a final concentration of 0.1 × 10−3 m and grown overnight at 18 °C. The cells were harvested by centrifugation (4000 rpm at 4 °C for 20 min) and resuspended in column buffer (20 × 10−3 m Tris-HCl, 200 × 10−3 m NaCl, 1 × 10−3 m EDTA, pH 7.4), followed by sonication (225 W, duty cycle 50%, VC 750, Sonics & Materials Inc., Newtown, CT) in an ice bath. The sonicated suspension was centrifuged at 4000 rpm for 20 min, and the supernatant was mixed with amylose resin and incubated for 20 min with gentle shaking. The mixture was then transferred to a purification column. After washing the resin with ample volume of column buffer, the ZFP was eluted with elution buffer containing 10 × 10−3 m maltose. Purified ZFPs were confirmed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure S6, Supporting Information).

Preparation of dsDNA Containing ZFP Binding Sites: Two types of dsDNAs with and without ZFP binding sites were prepared. Briefly, dsDNA with two ZFP binding sites was constructed by inserting two 20-base ZFP binding sequences at both end of gfp (green fluorescent protein) gene (700 bp) residing in pMAL-c2x vector through overhang PCR with a primer set (5′-CAC CAA GCT TTT AGC TTC CG-3 and 5′-TGC ATC TAG AGG ATT TGC TAA GG-3′) having HindIII and XbaI restriction enzyme sites, respectively. The resulting overhang PCR product was inserted into the corresponding multiple cloning site of pUC18a(+) vector. After that, 1152 bp of the dsDNA harboring two ZFP binding sites was prepared by PCR using a primer set (5′-ATT GTA CTG AGA GTG CAC CAT-3′ and 5′-TGA GCG CAA CGC AAT TAA-3′). Double-stranded DNA (991 bp) without ZFP binding sites was PCR-amplified from fimA gene (Klebsiella pneumoniae KTCT 2242) using a primer set (5′-ATG AAA ATC AAA ACA CTG G-3′ and 5′-TTA CTC GTA CTG CAC TTT GAA-3′).

Nanopore Experiments: Nanopores with a diameter of 4–5 nm were fabricated on suspended SiN membrane using TEM following the method reported earlier.[15] To reduce the dielectric noise, the front side

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of chip excluding the window region was passivated by hand-painted thin PDMS layer.[38] The nanopore chip was fixed in between two custom-built Teflon chambers that were filled with 20 µL of the electrolyte solution containing 1 m KCl and TE buffer (10 × 10−3 m Tris, 1 × 10−3 m EDTA, pH 8.0). Ag/AgCl electrodes were installed in cis and trans chambers. To analyze the characteristics of the IL-treated DNA using nanopore, DNA and each aqueous ILs were incubated in 1 m KCl to a final concentration of 150 × 10−12 m and 100 × 10−3 m, respectively, at room temperature for 1 h, which was then introduced into the cis chamber of the setup. To investigate the effect of IL on DNA–ZFP, DNA and ZFP with a molar ratio of 1:150 were preincubated for 1 h at room temperature in binding buffer (200 × 10−3 m Tris-HCl pH 7.0, 0.2 × 10−3 m of ZnSO4, 10 × 10−3 m MgCl2, 5 × 10−3 m DTT, distilled water, DNA 1.5 × 10−9 m). The reaction solution was then mixed with IL to a final concentration of 100 × 10−3 m and incubated further for 1 h in room temperature. The DNA–ZFP treated with IL was analyzed under the bias voltage of 150 mV. Electrical measurements were carried out using an Axopatch 200B and Digidata 1440A data digitizer (Molecular Devices, Sunnyvale, CA) at a sampling rate of 100 kHz. All data were analyzed with Clampfit 10.2 software (Molecular Devices).

AFM Analysis: To examine the structural conformation of IL-treated DNA, λ-DNAs (Bioneer, Daejeon, Korea) with and without ILs were analyzed by AFM. 1.5 × 10−9 m of λ-DNA was mixed with 100 × 10−3 m of ILs in 5 × 10−3 m HEPES (pH 8.0) with 10 × 10−3 m NiCl2 and incubated for 30 min at room temperature. High-grade mica discs (Ted Pella, Tustin, CA) were then put into the DNA solution for 30 min to induce divalent cation mediated DNA–mica binding. The chip was then washed with distilled water and dried under a gentle stream of nitrogen gas before AFM analysis (XE-70, Park Systems, Suwon, Korea). A noncontact cantilever probe (PPP-NCHR, Park Systems) was used to image the structural conformation of λ-DNA and IL-treated λ-DNA. All the AFM imagings were carried out with 0.3 Hz scan rate by tapping mode.

Fluorescence Microscopy: The λ-DNA was stained with SYBR Green to observe the structure of DNA–ILs complex in aqueous solution. λ-DNA was mixed with 100 × 10−3 m of ILs in TE buffer (10 × 10−3 m Tris, 1 × 10−3 m EDTA, pH 8.0) with 1× SYBR Green and incubated at room temperature for 30 min, which was then examined using fluorescence microscope (TE2000-U, Nikon, Tokyo, Japan). The λ-DNA without ILs was also examined as a control. The DNA molecules stained with SYBR Green were visualized with excitation and emission wavelength of 498 and 522 nm, respectively. All samples were kept in dark before observation to prevent the photobleaching of fluorescent dye.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgementsK.-B.J. and K.L. contributed equally to this work. This research was supported by the Pioneer Research Center Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (2012-0009575 and 2012-0009563), and the National Research Foundation of Korea (NRF-2012M3A7B4049864), and Global Ph.D. Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017H1A2A1046419).

Conflict of InterestThe authors declare no conflict of interest.

KeywordsDNA folding, DNA–protein interactions, ionic liquids, nanopores, signal enhancement

Received: April 10, 2018Revised: May 28, 2018

Published online: July 3, 2018

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