Letter pubs.acs.org/NanoLett
Voltage-Dependent Properties of DNA Origami Nanopores Silvia Hernández-Ainsa, Karolis Misiunas, Vivek V. Thacker, Elisa A. Hemmig, and Ulrich F. Keyser* Cavendish Laboratory, Department of Physics, University of Cambridge, JJ Thomson Avenue, Cambridge CB3 0HE, United Kingdom S Supporting Information *
ABSTRACT: We show DNA origami nanopores that respond to high voltages by a change in conformation on glass nanocapillaries. Our DNA origami nanopores are voltage sensitive as two distinct states are found as a function of the applied voltage. We suggest that the origin of these states is a mechanical distortion of the DNA origami. A simple model predicts the voltage dependence of the structural change. We show that our responsive DNA origami nanopores can be used to lower the frequency of DNA translocation by 1 order of magnitude. KEYWORDS: DNA origami, responsive materials, nanopores, nanotechnology
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their utilization as sensors capable of distinguishing between different ssDNA sequences.6,8 On the other hand, DNA has also been utilized to form responsive nanomachines that undergo motion. This movement has been triggered by different stimuli such as strands hybridization13−15 pH,16,17 enzymes,18 light,19 ions,20 and electric field.21 However, the utilization of DNA to build responsive nanopores has yet to be demonstrated. DNA origami possesses an intrinsic property that makes it sensitive to voltage, the negatively charged phosphate backbone of the DNA. In this Letter, we investigate the voltagedependent properties of a hybrid DNA origami nanopore based on a DNA origami nanopore with a long double-stranded DNA leash combined with glass nanocapillaries. We show that the hybrid DNA origami nanopores change under applied voltages and demonstrate their ability to control the rate of biomolecular translocations. Scaffolded DNA origami structures were constructed using the open source DNA origami software caDNAno.22 As schematically shown in Figure 1a, our DNA origami nanopore consisted of a flat 60 nm × 54 nm rectangular sheet two helices thick with a nanopore of 14 nm × 15 nm in the center and with a leash of double stranded DNA protruding from the central nanopore of approximately 450 nm length. In our design, double-helical DNA domains were packed in a square lattice.23 To ensure correct chemical connectivity of the leash to the sheet section we designed the DNA origami structure so that the leash was part of the scaffold and it was complemented with staples to form a double stranded helix. The DNA origami nanopore was constructed with the 8634 nt-long m13mp18-
ipid membranes in living systems possess nanopores that are responsible for the regulation of ionic transport and biomolecular trafficking. Some of these nanopores exert control over the passage of molecules, mediated by chemical and physical stimuli. These controlled processes are very important for the correct development of several physiological functions essential to preserve life such as energy production or protein synthesis.1 Artificial design of functional devices that resemble biological nanopores is attracting enormous interest in nanobiotechnology because such abiotic systems could enable us to gain insight in understanding the activity and mechanisms occurring in their natural counterparts. Most of these responsive artificial nanopores have been based on the incorporation of active molecules by chemical modification of the surface of solid-state nanopores such as polymer-based or silicon-based nanopores.2 In the last few years, the capabilities of DNA as a nanoscale building material have been vastly explored and multiple structures with accurate shape and dimensions have been constructed mainly utilizing the scaffolded DNA origami method.3 In this technique, a long single-stranded DNA (ssDNA) “scaffold strand” is folded into complex shapes aided by hundreds of short ssDNA strands (“staple strands”) in a process governed by the specificity of the base-pairs interaction.4 Recently, DNA nanotechnology has enabled the preparation of customized 2D and 3D structures containing a nanometer-sized pore that when combined with solid-state nanopores5−8 or embedded in a lipid bilayer9−11 have shown excellent properties as hybrid nanopores. These DNA origami nanopores present significant improvements for biomolecular sensing purposes.12 For instance, the ability to precisely tailor their pore size has enabled control over the folding of translocating dsDNA.8 Also the possibility to attach chemical residues within the pore with atomic precision has allowed for © XXXX American Chemical Society
Received: November 11, 2013 Revised: January 27, 2014
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All ionic current measurements were made using an Axopatch 200B (Axon Instruments, U.S.A.) amplifier in voltage-clamp mode. The signals were digitized with a NIPCIe- 6251 card (National Instruments). Data recording and analysis were performed with a custom program written in LabVIEW (LabVIEW 8.6, National Instruments). The formation of the hybrid DNA origami nanopore was performed by voltage-driven trapping onto the glass nanocapillary.5−8 DNA origami was added to the reservoir containing the nanocapillary tip to a final concentration of ∼0.5 nM in 1 M KCl, 5.5 mM MgCl2 solution buffered with 0.5 × TBE (pH∼8.2). A positive voltage applied in the back reservoir trapped the DNA origami nanopores onto the nanocapillary tip and was detected by the characteristic drop in ionic current (blue trace, Figure 2a). As described
Figure 1. (a) Schematic representation of the DNA origami structure with a rectangular sheet of dimensions of 60 nm × 54 nm, a central 14 nm × 15 nm nanopore and a double stranded leash of approximately 450 nm length. (b,c) Topographic AFM images of assembled DNA origami structures deposited on mica and imaged in air. Scale bar: 500 nm. The sheet and the leash are clearly distinguishable in panel c, whereas the central nanopore is better observed in panel b. (d) Schematic representation of the nanocapillary cells employed for the ionic current measurements. The nanocapillary is assembled in a PDMS mold connecting two fluid reservoirs that contain the measurement solution (1 M KCl, 5.5 mM MgCl2 solution buffered with 0.5 × TBE). Ag/AgCl electrodes are inserted in both reservoirs and connected to the amplifier. Samples are added to the reservoir containing the tip.
Figure 2. (a,b) Typical ionic current traces and schematic representations of the trapping of the DNA origami structure on the glass nanocapillary upon applying 400 mV showing two types of drop in current: low drop (blue trace, a) and high drop (red trace, b). (c) Histograms of the percentage drop in ionic current produced by the DNA origami nanopore trapping events on a single nanocapillary at different voltages. First population (lower drop in current) is marked with a blue bar and second population (higher drop in current) is marked with a red bar (colors are used to guide the eye). The area of the bars represents the amount of both populations. Resistance of this nanocapillary was 65 MΩ.
based scaffold.24 Of the 8634 bases of the scaffold, 7290 bases constituted the sheet hybridized with 198 staples and the other 1344 bases formed the leash with 39 complementary staples (see Supporting Information). DNA origami assembly was achieved by mixing scaffold and staples to a final concentration of 10 and 100 nM, respectively, in a 14 mM MgCl2 solution buffered with 0.5 × TBE (pH ∼ 8.2) and subjecting the mixture to thermal-annealing cycles for 23 h. The assembled structures were purified from the excess staple strands by centrifugation with 100 kDa MWCO filters. Correct assembly of the purified DNA origami structures was verified with atomic force microscopy (AFM). All three elements of the DNA origami structures, sheet, nanopore, and leash can be clearly distinguished in the AFM images shown in Figure 1b,c. Agarose gel electrophoresis experiments revealed the absence of aggregates during the assembly process (see Figure S1 in Supporting Information). Glass nanocapillaries were produced by pulling glass capillaries with a laser assisted pipet puller. Scanning electron microscope (SEM) images allowed us to estimate outer diameters of 41 ± 5 nm for these pulled nanocapillaries (see Figure S2 in Supporting Information). Nanocapillaries were assembled in polydimethylsiloxane (PDMS) cells as schematically shown in Figure 1d following a previously described procedure.25−28
previously,8 ejection of the DNA origami structure was achieved by reversing the voltage to −1000 mV, which enabled recovering the ionic current level corresponding to the bare nanocapillary. We performed such trapping and ejection multiple times in the same glass nanocapillary. Depending on the applied voltage, we observe two different types of decrease in ionic current upon trapping of DNA origami nanopores (compare the smaller drop in current in the blue trace, Figure 2a, and the bigger drop in current in the red trace, Figure 2b). To further investigate this feature, we repeatedly performed the trapping and ejection of the DNA origami structures at different voltages and with a range of nanocapillaries. An example of the histograms of the different values of the drops in ionic current detected for different voltages is shown in Figure 2c. We observe that at low voltage (300 mV), values are mainly distributed in a single population (marked in blue). Similarly, origami nanopores trapped at a high voltage (800 mV) also produced predominantly a single distribution (marked in red) but with a significantly bigger value than the observed at 300 mV. Interestingly, for intermediate voltages we observe two populations whose values correspond to those obtained for the B
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low (marked in blue) and high voltages (marked in red), respectively. Our data show an evolution of both populations as a function of the voltage: the higher the voltage, the greater the relative number of trappings that show a higher drop in ionic current (see also Figures S3−S6 in Supporting Information). DNA origami nanopore trappings are observed in the range of 300−800 mV in this nanocapillary. For lower voltages of 200 mV and below, we observe short ionic current blockades that we identify as transient trapping attempts of DNA origami (see Figure S7a in Supporting Information). On the other hand, at high voltages of around 1000 mV some complete DNA origami translocations through the pore can be occasionally detected (see Figure S7b in Supporting Information). This latter effect has been recently reported for DNA origami plates on silicon nitride nanopores.29 The absence of aggregates (see Figure 1b,c and Figure S1 in Supporting Information), which would produce a higher drop in ionic current, led us to speculate that more than one DNA origami conformation is possible during the trapping process. We studied the distribution dependence with voltage using an analogous DNA origami nanopore design without leash (see Figure S8 in Supporting Information). But in this case, only a single peak was observed for the whole voltage range (see Figure S9−S12 in Supporting Information). Therefore, we assign this voltage dependent trapping to the presence of the leash in the DNA origami design. We suggest that these two ionic current levels correspond to two conformations produced by a mechanical distortion of the DNA origami nanopore due to the electrophoretic force on the leash within the nanocapillary. Our results clearly prove that voltage plays an important role in the trapping of the DNA origami nanopore and consequently in its mechanical stability. We propose that the conformation leading to the first peak in the distribution (lower value, marked in blue) is the one with the DNA origami trapped with the sheet section flat but fluctuating on the nanocapillary tip and thus not completely sealing it (Figure 3a,b). In the following, we will call this the “initial conformation”. The second conformation corresponds to the DNA origami trapped with the sheet section buckled and partially introduced inside the nanocapillary due to the large electrophoretic force on the leash (Figure 3c,d), the “buckled conformation” in the following. We model our DNA origami structure as a thin elastic sheet supported by a circular boundary that represents the glass nanocapillary. The force deforming the sheet is assumed to be applied around the center since the leash is attached at that point. This force is also responsible for pulling the DNA origami onto the nanocapillary. This configuration of a stressed DNA origami sheet on a nanopore can be described with an analytical solution30 (see Supporting Information). It is known that the force F on the leash is proportional to the applied electric potential V.31 We suggest that the DNA sheet can plastically deform when the applied stress exceeds a certain value σt. The state with only elastic deformation corresponds to the DNA origami nanopore in the initial conformation (Figure 3a,b), whereas the plastic deformation leads to the buckled conformation (Figure 3c,d). We can estimate this stress σt if we determine V0, voltage required for obtaining an equal proportion of both conformations (initial and buckled), based on the experimental data. V0 is therefore used as an indicator of the applied stress. According to our model, V0 depends linearly on the resistance of the nanocapillary R with a scaling factor that includes the constant σt value (see eqs S5 and S6 in
Figure 3. Simplified schematic representations of the two proposed conformations of the DNA origami nanopore depending on the applied voltage once trapped onto the glass nanocapillary. (a) Perspective view and (b) side view of the initial conformation: the sheet section remains flat and fluctuates on the glass nanocapillary. (c) Perspective view and (d) side view of the buckled conformation: the sheet section is partially introduced in the glass nanocapillary. (e) Voltage V0 at which initial and buckled conformation are observed with about equal proportion as a function of the resistance of the nanocapillary. The red line represents a lineal fit to the experimental data. Data correspond to 12 experiments done in different nanocapillaries.
Supporting Information).30 As shown by the data and fit in Figure 3e, our result supports this interpretation. Thus, bigger nanocapillary resistances require higher voltages to observe both conformations with the same likelihood. This result evidence that the electric field pulling on the leash strand is leading to a force that may induce a plastic deformation of the DNA origami nanopore. As the model predicts the voltage dependence and we also observe an increased ionic current blockage in the buckled conformation, we tested the possibility to mechanically control the passage of molecules depending on the DNA origami conformation. For this purpose, linear double-stranded λ-DNA (48.5 kbp) was used to investigate the translocation processes. C
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origami buckled conformation, which could explain the decrease in the translocations frequency. In conclusion, we present here the first experimental evidence of the formation of hybrid DNA origami nanopores that change their conformation as a response to the applied voltage. We have shown that voltage has a pronounced effect on the conductance of the DNA origami nanopore that we assign to the mechanical distortion produced by the presence of a double stranded DNA leash in the DNA origami structure. The mechanical distortion can be explained in terms of a simple model for a supported DNA sheet. We have also proved the ability of these DNA origami nanopores to control the frequency of λ-DNA translocation. Our results opens the possibility to utilize these artificial DNA origami structures as smart nanopores whose behavior can be controlled with an external stimulus.
Initially, hybrid DNA origami nanopores were formed by applying positive voltage as previously described. λ-DNA was then added to the same reservoir containing the DNA origami nanopores to a final concentration of ∼1.5 nM. Upon applying positive voltage, λ-DNA translocates through the hybrid DNA origami nanopore5,8 that, depending on the voltage, is either in the initial or buckled conformation. In order to record λ-DNA translocations through the bare nanocapillary, we applied negative voltage to eject the DNA origami nanopore and recover the nanocapillary tip and then positive voltages to get λDNA translocations until a new DNA origami nanopore was trapped. Figure 4a shows some examples of the ionic current
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ASSOCIATED CONTENT
S Supporting Information *
SEM image of glass nanocapillary and histogram of the outer diameter, agarose gel of purified DNA origami structure, schematic representation of the analogous DNA origami structure without leash, further examples of the histograms of the percentage drop in ionic current, further details about the model, ionic current events showing attempt of trapping and translocation of DNA origami, ionic current traces and histograms of the λ-DNA translocations, staple strands sequences, and scaffold-staple layout for the DNA origami structure. This material is available free of charge via the Internet at http://pubs.acs.org.
Figure 4. (a) Typical λ-DNA translocations events for the nanocapillary (black trace), hybrid DNA origami nanopore in the initial conformation (blue trace), and the buckled conformation (red trace) recorded at 400 mV. (b) Bar chart of normalized frequency of the λDNA translocations through the nanocapillary (black bar) and the hybrid DNA origami nanopore in the initial (blue bar) and in the buckled conformation (red bar) at 300, 400, and 500 mV of applied voltage. For comparison of the data, normalization was done with respect to the frequency obtained in case of the bare nanocapillary. Error bars correspond to the standard deviation of the values averaged from three independent experiments. Resistance of this nanocapillary was 59 MΩ.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
blockade events observed due to λ-DNA translocations through the nanocapillary (black trace), through the hybrid DNA origami nanopore in its initial conformation (blue trace) and through the hybrid DNA origami nanopore in its buckled conformation (red trace) at 400 mV. We calculated the λ-DNA translocation frequency for each case: the bare nanocapillary, the hybrid DNA origami nanopore in the initial conformation, and the hybrid DNA origami nanopore in the buckled conformation. For each experiment, the concentration of λ-DNA was taken into account. The translocation frequency was calculated dividing the number of total events by the total time of ionic current recording for each of the three cases. The calculated frequency was then normalized by the λ-DNA concentration and averaged over three different experiments. Figure 4b shows the normalized frequency of λ-DNA translocations detected in the hybrid DNA origami nanopore in its initial conformation (blue bar) and its buckled conformation (red bar) with respect to the nanocapillary one (black bar) at 300, 400, and 500 mV. For all voltages, we observed an approximately 10-fold decrease in the translocation frequency between nanocapillary and the hybrid DNA origami nanopore in its initial conformation and also a further 10-fold decrease for the buckled conformation. We assume that the central region is axially elongated and therefore the effective size of the nanopore is decreased in the DNA
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Nicholas Bell for help with SEM imaging and for helpful discussions and Tim Liedl for help with DNA origami design. S.H.A. and U.F.K. acknowledge support from an ERC starting grant and from Oxford Nanopore Technologies® (www.nanoporetech.com). K.M. was supported by the EPSRC program. V.V.T. acknowledges funding from the Cambridge Commonwealth Trust, the Jawaharlal Nehru Memorial Trust, and the Emmy Noether program of the Deutsche Forschungsgemeinschaft (DFG). E.A.H. acknowledges support from Schweizerische Studienstiftung.
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