A dynamically gated triangular DNA nanopore for molecular sensing and cross-membrane transport

doi:10.21203/rs.3.rs-3878148/v1

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A dynamically gated triangular DNA nanopore for molecular sensing and cross-membrane transport

https://doi.org/10.21203/rs.3.rs-3878148/v1

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published 22 Aug, 2024

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Synthetic membrane nanopores made of DNA are promising systems to sense and control molecular transport in biosensing, sequencing, and synthetic cells. Dynamically gating cargo transport like the natural ion channels and systematically increasing the lumen size have become long-standing desires in developing nanopores. Here, we design a triangular DNA nanopore with a large dynamically-gated lumen. It can switch between expanded and contracted states without changing its stable triangular shape, whereby specific DNA bindings as stimuli mechanically pinch and release the three corners of the triangular frame. Transmission electron microscopy images and molecular dynamics simulations illustrated the large lumen up to 539 nm², the stable architectures, and the high shape retention. Single-channel current recordings and fluorescence influx studies demonstrated the low-noise repeatable readouts and the controllable cross-membrane macromolecular transport. We envision that the proposed DNA nanopores could offer powerful tools in molecular sensing, drug delivery, and the creation of synthetic cells.

Biological sciences/Biotechnology/Nanobiotechnology/Nanopores

Physical sciences/Nanoscience and technology/DNA nanotechnology/DNA nanomachines

Membrane nanopores^1–3, the hollow barrels spanning biological or synthetic membranes, play vital roles in various bodily functions and molecular biology including signal transduction⁴, cross-membrane molecular transport⁵, creating synthetic cells^6,7, and label-free molecular sensing^8,9. Among the known nanopores in nature, ion channels are the most studied and functionally complex pores with two important properties: highly selective filter and mechanically gated transport^10–12. Responding to environmental stimuli, ion channels can dynamically change their inner width of a few nanometers to allow or prevent the passage of cargo. Next-generation nanopores mimicking the dynamic gating behaviors of the natural ion channels have attracted considerable scientific and technological interest; however, existing two challenges are the dynamically gated pore with a much wider lumen for controllable cross-membrane macromolecular transport and the structurally stable pore for low-noise repeatable electrical readouts in molecular sensing.

The rational route to next-generation synthetic nanopores could be DNA nanotechnology, an ideal tool for building large de novo architectures^13,14. With the strict base-pairing rule and highly predictable precise structures, DNA nanotechnology enables the programmed production and rapid optimization of multifunctional DNA nanostructures^15–18. Moreover, achieved applications of the DNA nanostructures indicate that the stability^19,20 of synthetic nanostructures determines their feasibility in a variety of applications such as precision measurement tools^21–23 and smart drug carriers^24–26. Thus, numerous studies adopt stable triangular architectures or variants^27,28, including triangular subunits, and advanced molecular dynamics simulations are applied to validate the stability of the designed DNA nanostructures^29–31. Nanopores entirely engineered by DNA nanotechnology have been previously reported^32–38, however, stimuli-responsive large DNA nanopores with dynamically controlled lumen sizes and low-noise repeatable electrical readouts remain challenges. Nevertheless, the ease of de novo design of nanopores with DNA is apparent.

Existing DNA nanopores are mostly built based on the parallel alignment of the DNA duplexes; however, the disadvantages are the reported narrow lumen and difficulty in accommodating the gating mechanism or other functional units. The very recently pioneered DNA nanopores overcome these limitations by arranging the bundled DNA duplexes parallel to the membrane^39,40. By controlling the length of the nanopore subunits formed by bundled DNA duplexes, the achieved lumen areas range from tens to hundreds of nm², which is one to two orders of magnitude larger than typical protein pores³⁹. The self-assembled numerous DNA duplexes in the same plane form a broad plate to accommodate wide lumen, conformational transition mechanisms, stimuli-responsive units, and other functional units⁴⁰. Nevertheless, structural stability has not been considered in designing these DNA nanopore designs. In the lumen-tunable static nanopore design³⁹, unstable DNA architecture may lead to lumen size and shape changes, further influencing the precise sensing and controlled transport of protein cargoes. In the proposed stimuli-responsive DNA nanopore⁴⁰, the dynamic conformational transitions are actuated by the mechanical shape change of the quadrilateral frames, while the most known natural ion channels open and close like a diaphragm¹⁰. The shape change may allow the relatively larger molecules with matched cross-sectional shapes to pass through the constricted quadrilateral nanopores. DNA nanopores with fixed shapes could use a uniform electrical model to elucidate the passage of molecules and easily predict the electrical readouts. Moreover, the structural flux of the semiflexible quadrilateral frames would cause unrepeatable electrical readouts and significant signal noise.

Here, targeting the controllable transport of macromolecules and low-noise repeatable electrical readouts in molecular sensing, we develop a structurally stable triangular DNA nanopore of considerable size and realize mechanical lumen transition without altering its shape. Three pore subunits assembled from side-by-side bundled DNA duplexes are linked to form a triangular frame with a wide lumen. Specific DNA bindings are used to pinch and release the three corners of the triangular frame, thereby mechanically activating the DNA nanopore to switch between the DNA nanopore-expanded state (DNP-E) and the DNA nanopore-contracted state (DNP-C). Rather than that the very recently pioneered DNA nanopores realize the gated transport by equipping flap-like gates¹⁴ or changing the shape of the semi-flexible quadrilateral frames⁴⁰, the proposed nanopores do not undergo any shape changes when switching between expanded and contracted states. It avoids the passage of relatively larger molecules with matching cross-sectional shapes through the constricted quadrilateral nanopores. This property ideally mimics the diaphragm-like lumen transition of natural ion channels and could significantly contribute to structural stability, precise gated transport, and repeatable low-noise signals in electrical sensing. We anticipate that the suggested DNA nanopores could offer valuable insights for developing and applying the next-generation nanopores.

Triangular DNA nanopore

In response to existing challenges in developing next-generation nanopores, we have designed a DNA nanotechnology-engineered triangular nanopore with a dynamically gated lumen for sensing and controlling molecular transport. The triangular DNA nanopore is composed of three subunits interconnected by single-stranded hinges (Fig. 1a). Each subunit adopts a sandwich structure, where the side-by-side bundled long DNA duplexes act as the core layer, and the bundled short DNA duplexes serve as the inner and outer layers. The three-layer middle part of the pore subunit serves as the solid girder and two neighboring single-layer ends interconnected by the single-stranded hinge form a v-clip. Triggered by specific DNA bindings, the three v-clips can be pinched and released to realize the bidirectional mechanical transition of the triangular DNA nanopore. In particular, nine single-stranded DNA (referred to as Triggers) are employed to pinch the three corners of the large triangular frame simultaneously and contract the lumen. Conversely, targeting switching from DNP-C to DNP-E, precisely defined twelve single-stranded DNA (referred to as Reverse triggers) are utilized to replace the positive triggers and release the three corners of the DNP-C. It is even more noteworthy that, in the mechanical transition of the designed nanopore, the lumen retains the stable triangular shape at both contracted and expanded states.

According to the route designs of the triangular DNA nanopores in different states (Supplementary Figs. 1 and 2), DNP-E and DNP-C were assembled by annealing the scaffold DNA and the multifunctional staple strands (Supplementary Data 1). In the upper region of the representative gel image (Supplementary Fig. 3), homogeneous bands of DNP-E and DNP-C presented different mobilities during gel electrophoresis, providing evidence of the formation of singular assembly products for both DNP-E and DNP-C. After the purification of the assembled products by ultrafiltration units, different morphologies (Fig. 1b-g) of DNP-C and DNP-E were observed by negative-stained transmission electron microscopy (TEM).

In line with the design concept, the triangular DNA nanopores in different states have different lumen sizes (Fig. 1b-g), as shown for DNP-E and DNP-C with the cross-sectional area of 539.0 ± 120.2 nm² (± SD, n = 20) and 237.9 ± 77.3 nm² (± SD, n = 20), respectively. Relative area analyses showed that the lumen area of DNP-E contracted upon the induction of triggers, and the lumen area of DNP-C expanded through the treatment of reverse triggers (Fig. 1c, f). To investigate whether lumen shape change accompanied the mechanical transition of the proposed nanopores, we counted the angular distribution of DNA nanopores in different states. We found that additional trigger or reverse trigger treatments had no significant effect on the θ angle (relative to the bottom subunit) of DNA nanopores (Fig. 1d, g), which mostly stayed around 60º. These data show that the triangular DNA nanopores can be dynamically gated without undergoing notable lumen shape changes.

To facilitate embedding the proposed DNA nanopores into lipid bilayers, we utilized twenty-one handles of staple strands to hybridize the cholesterol strands (Fig. 1h). Cholesterol-modified DNP-E and DNP-C showed upshifted bands in gel electrophoresis analysis (Supplementary Fig. 4), demonstrating that the cholesterol strands were attached to the DNA nanopores as designed. Small unilamellar vesicles (SUVs) were used to investigate the interaction between cholesterol-tagged DNA nanopores and lipid bilayers (Supplementary Fig. 5a). The signal intensity of the upshifted bands in DNP-C samples gradually increased as the lipid concentration of SUVs increased (Supplementary Fig. 5b). In contrast, no upshifted bands were observed for DNP-C samples in the absence of SUVs. The signal intensity analysis revealed that the upshifted bands were caused by the interaction between DNP-C and SUVs (Supplementary Fig. 5c). In addition to gel electrophoresis, TEM images confirmed the binding of synthetic DNA nanopores to SUVs. The distinctly shaded areas representing the different lumens of DNP-C and DNP-E indicated that these DNA nanopores interacted with and then inserted into the SUVs (Fig. 1i). Moreover, to visualize the interaction between cholesterol-tagged DNA nanopores and giant unilamellar vesicles (GUVs), we employed twelve additional handles in DNA nanopores to gather the Cy3 strands (Fig. 1h). Representative confocal images depicted the efficient binding of abundant Cy3-marked DNP-C to rhodamine-contained lipid membranes of GUVs, as evidenced by the high localization of the fluorescent signal between the green and red channels (Fig. 1j).

Electrical characterization

The insertion of cholesterol-tagged DNA nanopores into the lipid membrane (Fig. 1k) was confirmed by analyzing the conductance properties of DNP-C and DNP-E using single-channel current recordings. In particular, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was used to produce planar lipid bilayers. In every microelectrode cavity of the current recording chip, the electrolyte (1 M KCl, 10 mM HEPES, pH 7.4) was separated by a planar lipid bilayer into two chambers, including a cis side and a trans side. When a potential was applied across the lipid membrane, electrolyte ions were induced, ion flow occurred through the lumen of the embedded DNP-C (Fig. 2a), and a constant current of 0.61 nA was observed at an applied voltage of + 80 mV. In contrast, when the applied voltage was changed to − 80 mV, the recorded current was constant at − 0.62 nA. The single-channel current trace (Fig. 2b) indicated the successful insertion of DNP-C into the planar lipid bilayer. Statistical analysis of the conductance distribution of DNP-C showed the peak conductance with an average of 7.75 ± 0.34 nS (± SD, n = 22) (Fig. 2c). DNP-E has a larger lumen than DNP-C. Thus, the inserted DNP-E should allow more electrolyte ions to pass through its lumen than the inserted DNP-C. Consistent with the concept of both schematics (Fig. 2a, d), the constant current of DNP-E (1.26 nA) was higher than that of DNP-C (0.61 nA) when a voltage of + 80 mV was applied across the inserted DNP-E and DNP-C (Fig. 2b, e). The conductance peak histogram of inserted DNP-E appeared at 16.58 ± 0.54 nS (± SD, n = 20) (Fig. 2f), which further suggested that the lumen size of DNP-E was larger than that of DNP-C. The relatively more concentrated distribution of the peak conductance of DNP-C and DNP-E illustrated the repeatable electrical readouts brought by the structurally stable triangular lumen. Moreover, control experiments of DNP-C and DNP-E without cholesterol modification showed no measurable ionic currents (Supplementary Fig. 6), thus verifying that cholesterol modification is essential for inserting triangular DNA nanopores into planar lipid bilayers. Successful insertion of cholesterol-labeled DNP-C and DNP-E into lipid membranes was also verified by linear current-voltage relationships with different slopes (Supplementary Fig. 7), as expected for vertically symmetric DNA nanopores.

To further explore the dynamic transition of triangular DNA nanopores between expanded and contracted states, we separately added triggers and reverse triggers on the cis side of membrane-spanning DNP-C and DNP-E (Fig. 2g). A representative current trace indicated that the current signals of inserted DNP-C changed from 0.63 nA to 1.07 nA with a constant voltage of + 80 mV (Fig. 2h), reflecting the lumen expansion induced by reverse triggers. Considering the conductance properties of DNP-C and DNP-E, the initial and terminal current signals of the representative current trace (Fig. 2h) implied that the transition of DNA nanopores from DNP-C to DNP-E was realized. In the power spectrum analysis (Fig. 2i), the noise profile of the DNP-C processed with reverse triggers was higher than before, denoting that more fluctuations were generated in the DNP-C treated with reverse triggers than before. After treating the membrane-spanning DNP-E with triggers, the recorded current signal changed from 1.20 nA to 0.73 nA (Fig. 2j), suggesting that the transition of DNA nanopores from DNP-E to DNP-C was fulfilled. Interestingly, the dynamic transition from DNP-E to DNP-C took less than 2 s (Fig. 2j), which was much shorter than the switching duration from DNP-C to DNP-E (Fig. 2h). The lateral membrane pressure might accelerate the contracting process of the inserted DNA nanopore. Moreover, we noticed the achieved low-noise signal, which could be credited to the stable triangular lumen. Compared with inserted DNP-E without any treatment, triggers-treated DNP-E reduced power spectral noise (Fig. 2k), indicating a reduction in the lumen size. To examine the specificity of the triggers and reverse triggers that activate conformational transitions of DNA nanopores in different states, random strands (Supplementary Data 1) were separately introduced on the cis side of membrane-spanning DNP-C and DNP-E. Single-channel current recordings of random strands-incubated DNA nanopores showed no apparent fluctuating signals (Supplementary Fig. 8), further emphasizing the importance of triggers and reverse triggers for dynamic switching between DNP-C and DNP-E. Repetitive current signals demonstrated that the majority of DNP-C expanded the pore lumen in response to reverse triggers, and DNP-E contracted the pore lumen in the presence of triggers (Supplementary Fig. 9). This further validates the feasibility of the bidirectional mechanical transition design for the triangular DNA nanopore.

Computational analysis

To assess the structural stability of triangular DNA nanopores in different states, we characterized two different DNA nanopore designs by oxDNA^41–43 simulations. From a macro-perspective, statistical analyses of the relative angles in both DNA nanopore designs demonstrated a more pronounced fluctuation in the expanded DNA nanopore compared to the contracted one (Fig. 3a, b). The relative areas of the two DNA nanopore designs did not change significantly during the molecular dynamics simulations, indicating that their distinct pore lumens can be effectively maintained. DNP-E exhibited a notably higher root-mean-square deviation (RMSD) as compared to DNP-C, predominantly due to the elongated core layers positioned at the triangulated nanopore corners (Fig. 3c). In previous oxDNA simulations, the phenomenon of splaying at the ends of origami structures has been documented⁴⁴. However, whether this occurrence represents an experimental phenomenon or a simulation artifact remains an unresolved inquiry. The tight packing of the helices at the corners of DNP-C effectively suppresses this splaying behavior of lumen-facing DNA strands, whereas DNP-E, owing to its relatively spacious arrangement, manifests more pronounced splaying. Despite the fact that the proportion of broken base pairs remained below 1.5% for both designs throughout the simulations (Fig. 3d), DNP-C displayed superior integrity at a finer scale compared to DNP-E.

The conductance of both DNA nanopore designs was initially computed using the simple cylinder model. The conductance ratio of DNP-E to DNP-C was 2.34, closely aligning with the experimental ratio value (2.14) derived from recorded ionic currents. Notably, membrane nanopores made with DNA inherently possess ionically leaky walls. To account for this factor, we employed the steric exclusion model (SEM)^45,46 to simulate the conductance of both designs based on 3D conductivity maps. Specifically, ten configurations each of DNP-C and DNP-E were selected (Supplementary Videos 1 and 2), mapped into an all-atom representation, and embedded within DPhPC lipid bilayers to allow the calculation of 3D conductivity maps (Supplementary Fig. 10a). The conductivity maps were calculated from the distance from lipid or DNA utilizing local conductivity functions specific to the lipid and DNA components (Supplementary Fig. 10b) under an applied voltage of 80 mV and 0.4 M KCl. Figure 3e depicts the transverse view (top) and side view (bottom) along the center of each system. The peak conductance distributions observed in the SEM data for the two DNA nanopore designs were notably higher than those calculated from the cylinder model (Fig. 3f). This difference was likely attributed to the careful consideration of significant ionic leakage among the inherent structures of DNA nanopores during the SEM simulations.

While experimental measurements showed a closely matched conductance ratio of DNP-E to DNP-C compared to values obtained from SEM simulations or cylinder model calculations, the observed absolute conductance values were significantly smaller than the corresponding theoretical estimates. This discrepancy aligned with findings from prior studies on large DNA nanopores^14,39. We hypothesize that the lower experimental conductivities of DNP-C and DNP-E compared with those of solid-state nanopores of similar internal dimensions⁴⁷ may be caused by the continuous compression of the surrounding lipid molecules³³ and structural distortions^48,49 induced by the applied voltages⁴⁰. Moreover, we note that, for a select few DNA nanopore systems of well-defined transmembrane pore geometry, a good agreement between simulated and experimentally measured conductance values was reported^50,51. Future molecular dynamics simulations incorporating interactions between lipids and DNA may reveal factors underlying these disparities. Nevertheless, oxDNA simulations provided evidence for the structural stability of the two DNA nanopore designs, confirming that triangular DNA nanopores in different states possess distinct stable lumens.

Protein sensing

The dynamically-gated large lumens of triangular DNA nanopores hold great potential in molecular sensing. Hence, we employed membrane-spanning DNP-C and DNP-E to explore whether designed DNA nanopores can sense the trypsin molecule (4.0 nm), a model protein with a net positive charge at pH 7.4. In a typical current trace recorded at a constant voltage of + 50 mV, the introduction of trypsin regularly blocked the ion flow of inserted DNP-C. Notably, an individual blockade was identified as a single event of trypsin translocation (Fig. 4a), which was characterized by the dwell time (T_off) and the blocking amplitude (A) from the open-channel amplitude (I_O). Two distinct types of trypsin translocation events in embedded DNP-C were identified by analyzing the percentage of blocking amplitude and dwell time (Fig. 4b, c). More than 50% of trypsin translocation events clustered at a blocking amplitude of 30.19% ± 2.74% (± SD, n = 35). The remaining events clustered at a blocking amplitude of 53.32% ± 2.26% (± SD, n = 34). Given the current-dependent nature of trypsin translocation on well-developed DNA nanopores^14,34, Type I events with partial blocking amplitudes represent transient interactions of positively charged trypsin with embedded DNP-C (Fig. 4d). Contrastingly, Type II events show that trypsin molecules are fully translocated across the whole lumen of embedded DNP-C (Fig. 4e). Intermittent interactions of positively charged trypsin molecules with negatively charged inner walls of DNA nanopores may prolong the dwell time of Type II events. Following this concept, a scatter plot of trypsin translocation events sensed by embedded DNP-C showed that the average dwell time of Type II events (0.69 s) was much longer than that of Type I events (0.04 s).

Additionally, membrane-spanning DNP-E was used to sense the trypsin molecules, which was analyzed by single-channel electrophysiological recordings. Based on the representative current trace of embedded DNP-E after the introduction of trypsin (Fig. 4f), trypsin translocation events with diverse blocking amplitudes and dwell times could be discovered. Interestingly, there were no clearly distinct clusters in the scatter plot (Fig. 4g). Notably, the trypsin translocation events recorded from embedded DNP-E exhibited a blocking amplitude of 28.13% ± 7.38% (± SD, n = 85), which was narrower than the blocking amplitudes of trypsin translocation events from embedded DNP-C. Given that DNP-E adopted a larger lumen size than DNP-C, we suggested that the majority of recorded trypsin translocated completely through the embedded DNP-E rather than interacting transiently with its lumen opening (Fig. 4h).

Following a consistent electrical model for both DNP-C and DNP-E, the blocking amplitude of full trypsin translocation sensed by DNP-C was expected to be 2.14-fold that of DNP-E, which was close to the 1.90-fold derived from experimental data (Fig. 4b, g), further validating the superiority of the proposed shape-fixed DNA nanopores in elucidating molecular translocations and predicting electrical readouts. The noise in the power spectra of both DNP-C and DNP-E was considerably weaker before the introduction of trypsin treatment than afterward (Supplementary Fig. 11), verifying that both DNA nanopore designs were capable of sensing trypsin translocation. Representative single-channel current traces (Fig. 4c, f) and corresponding scatter plots (Fig. 4b, g) indicate that DNP-C can sense the trypsin molecules with low-noise repeatable readouts compared to DNP-E. In more detail, DNP-C can distinguish two different types of trypsin translocation events, while DNP-E cannot. This suggests that maintaining a closer match between the lumen and the molecule contributes to more precise sensing in the triangular DNA nanopore.

Cross-membrane transport

Finally, we investigated whether the different lumens of triangular DNA nanopores allow the transmembrane transport of macromolecules of different sizes. In contrast to SUVs, GUVs provide a better model for simulating physiological cell membranes and can be used to explore the cross-membrane transport of specific macromolecules through the embedded triangular DNA nanopores. We added FITC-dextran (40 kDa, Radius ≈ 4.8 nm) and TRITC-dextran (500 kDa, Radius ≈ 15.9 nm) to the external solution of GUVs while incubating with cholesterol-free DNA nanopores. No significant increase in fluorescent signal was measured from the interior of GUVs incubated with cholesterol-free DNA nanopores (Supplementary Fig. 12 and Supplementary Videos 3 and 4), verifying that cholesterol-free DNA nanopores were unable to insert into lipid membranes, which was consistent with the data obtained from single-channel current recordings (Supplementary Fig. 6).

With the assistance of cholesterol moieties, membrane-spanning DNP-C allowed the influx of smaller FITC-dextran (40 kDa) into GUVs while preventing the influx of larger TRITC-dextran (500 kDa) (Fig. 5a, b and Supplementary Video 5). Representative confocal images and associated fluorescence traces demonstrated that both macromolecules can enter the interior of the corresponding GUVs through the transmembrane lumen of embedded DNP-E (Fig. 5c, d and Supplementary Video 6). After 3 h of incubation, 46.7% of GUVs showed FITC influx in the presence of DNP-C treatment, and only 3.7% of GUVs were simultaneously filled with TRITC-dextran (Fig. 5e). In the case of DNP-E treatment, more than 30% of GUVs displayed influx of both two macromolecules, indicating that the expanded DNA nanopore allowed for the simultaneous transport of FITC-dextran and TRITC-dextran (Fig. 5f). Collectively, these results confirm that cholesterol-modified triangular DNA nanopores with different lumens can spontaneously insert into the lipid membranes of GUVs. These findings demonstrate that both DNA nanopores can function as size-selective gateways to control the transmembrane transport of various molecules.

In this work, we develop a triangular DNA nanopore with a dynamically-gated lumen. The main advantages of the developed nanopore are the large enough lumen sizes to accommodate proteins, stable triangular structure made with DNA, and dynamic diaphragm-like lumen transition mimicking natural ion channels. Our design bundles the DNA duplexes into pore subunits and then interconnects three subunits to form a broad plate to provide a large lumen up to 539 nm². Numerous DNA structures adopting the stable triangular architectures^27,28 or the variants including triangular subunits realized advanced precision measurement at the nanoscale and targeted drug delivery, while structural stability has been neglected in the rapid development of the nanopores made with DNA. Here, the stable triangular shapes of the DNP-E and DNP-C significantly contribute to the achieved low-noise and repeatable electrical sensing. Differing from tuning the lumen area through shape change of the quadrilateral frames⁴⁰, our design essentially adjusts the side lengths of the triangular frame by pinching and releasing its three corners. The nanopore undergoes no shape change, preventing the relatively larger proteins with matched cross-sectional shapes from passing through the constricted quadrilateral nanopores. Moreover, the fixed triangular shape of the proposed nanopore during the mechanical transition enables us to use a uniform electrical model to elucidate the passage of molecules and easily predict the electric readouts. These advantages together make the suggested DNA nanopores a potential approach to the next-generation nanopores.

Proposed nanopores entirely engineered by DNA nanotechnology allow rational de novo design of complex structures. The end-to-end linked numerous DNA duplexes are arranged parallel to the membrane to form a broad frame capable of accommodating wide lumen, conformational transition mechanisms, stimuli-responsive units, and other functional units. The proposed nanopore design route could provide unprecedented freedom in selecting the sizes and shapes to match the analytes or applications. More pore subunits could be linked to form quadrilateral, hexagonal frames, and even subcircular nanopores. Here, we tend to choose the triangular frame because it could provide relatively higher structural stability; however, this point must be confirmed by more experimental results. The lengths of the subunits can also be changed to tune the pore sizes, and the lengths are not necessary to be equal. Thus, there is nearly no limit on the pore size and shape selection. Moreover, by adjusting the relative locations and lengths of the outer and inner layers to the core layers of the pore subunits, we could customize the mechanical contraction and expansion of the nanopore, which enables the mechanically gated transport of specific proteins. In the future, the theoretically highly programable sizes, shapes, and mechanical transitions provided by our unique nanopore design route would deliver a substantial change to the traditional approach of using static nanopores in molecular sensing and cross-membrane transport.

In conclusion, by mimicking the diaphragm-like transition of natural ion channels and adopting a stable triangular architecture, we have proposed a design of dynamically gated DNA nanopore with a large lumen for molecular sensing and controllable cross-membrane transport. It offers solutions to the two main challenges in developing nanopores, including dynamically gating cargo transport like natural ion channels and systematically increasing the lumen size. We envision that the proposed DNA nanopores could provide powerful tools in biosensing and synthetic biology.

Materials

M13mp18 single-stranded DNA was obtained from New England Biolabs. All oligonucleotides were purchased from Sangon Biotech. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE), DOPE-Cy5, and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) were procured from Avanti Polar Lipids. Unless otherwise stated, all other reagents and solvents were purchased from Sigma-Aldrich.

Preparation of DNA nanopore

DNA nanopores in both states were designed by using the honeycomb lattice version of CaDNAno software. Detailed route designs for both structures are shown in Supplementary Fig. 1. The concrete sequences of all used staple strands are shown in Supplementary Data 1. In a typical reaction, scaffold DNA (10 nM) was mixed with corresponding staple strands (50–100 nM) in TE-Mg²⁺ buffer (10 mM Tris-HCl, 1 mM EDTA, and 10 mM MgCl₂, pH 8.0). The mixture was then annealed with a LifeTouch thermal cycler (Bioer Technology, China). The DNA nanopores were assembled using a 24 h annealing procedure: initially, the mixture was cooled from 95℃ to 60℃ at a rate of 1℃ per 10 min, then to 25℃ at a rate of 1℃ per 30 min, and finally kept at 4℃ until ultrafiltration. After the annealing reaction, the synthetic DNA nanopores were ultrafiltered with Amicon Ultra-0.5 ml 100 kDa centrifugal filters to remove excess staple strands. The ultrafiltration was conducted at 2500 rpm for 3 min and repeated at least three times. In addition, cholesterol strands (5’-GATGCATAGAAGGAA-3’-Chol) were mixed with purified DNA nanopores at a ratio of two times relative to the total number of cholesterol attachment sites on the DNA nanopores and incubated at 37℃ for 12 h to produce cholesterol-tagged DNA nanopores. Optionally, Cy3 fluorophores can be attached to DNA nanopores by treating purified DNA nanopores with a 10-fold excess concentration of Cy3 strands (5’-GCTAGCATGCTG-3’-Cy3) at room temperature for 12 h. After additional modifications with cholesterol moieties and Cy3 fluorophores, the desired DNA nanopores were eventually obtained by multiple ultrafiltration.

Preparation of lipid vesicles

For SUVs, the DOPC solution (10 mg/mL in chloroform, 50 µL) was dried by argon flow in a glass vial (2 mL) to yield thin films. The obtained films were suspended by HEPES-Na⁺ buffer (50 mM HEPES, 500 mM NaCl, pH 7.4) and then sonicated for 30 min to generate homogeneous SUVs. Dynamic light scattering was performed using a Malvern analyzer (Zetasizer Nano) to determine the size distribution of the acquired SUVs. For GUVs, a lipid solution (DOPC/DOPE = 9.9:0.1, 10 mg/mL in chloroform, 10 µL) was loaded onto an indium tin oxide (ITO)-coated glass slide. After 5 min of evaporation, dried lipid films were formed. The glass slide carrying lipid films was first placed into a Vesicle-Pre-Pro device (Nanion Technologies, Germany). An O-ring was placed around the lipid films, and then sorbitol (1M, 300 µL) was added to the lipid films to fill the entire O-ring. Subsequently, another ITO glass slide was mounted on top of the O-ring to ensure that a sealed chamber was formed between the two ITO glass slides. Finally, an alternating electric field was applied for 2 h to produce GUVs. Notably, all obtained lipid vesicles (SUVs and GUVs) were stored at 4 ℃ and used within 48 h.

Agarose gel electrophoresis

For samples containing only DNA nanopores, the mobility of various samples was analyzed using agarose gel electrophoresis (1.0%, TE-Mg²⁺ buffer) at 4 ℃. For samples of cholesterol-tagged DNA nanopores, they were subjected to agarose gel electrophoresis (1.0%, PBS buffer). To study the interaction of cholesterol-tagged DNA nanopores and SUVs, equal amounts of DNP-C (5 nM) were incubated with a range of lipid concentrations of SUVs (0, 5, 10, 20, and 40 nM), respectively. After 1 h of incubation, the mixed samples were subjected to agarose gel electrophoresis (1.5%, HEPES-Na⁺ buffer). All agarose gels were premixed with 0.01% SYBR™ Safe (Invitrogen, USA) and run at 100 V for 60 min. All gel images were captured with a Tanon 4600SF Multifunctional Imaging System (Tanon, China).

TEM characterization

Purified DNA nanopores without cholesterol modification and SUVs treated with cholesterol-tagged DNA nanopores were characterized separately using negative-stain TEM. Briefly, diverse samples (2 µL) were adsorbed on glow-discharged, carbon-coated TEM copper grids for 5 min, respectively. Then, the remaining solution on these TEM grids was carefully removed with filter paper. These grids were then treated with a staining solution (1.0% uranyl acetate) for 30 sec. Immediately, the additional staining solution was absorbed with filter paper. Finally, TEM images of all samples were taken with a JEM 1400 Plus transmission electron microscope (JEOL, Japan) at 100 kV.

Single-channel current recordings

An integrated chip-based parallel bilayer recording device (Orbit Mini, Nanion Technologies, Germany) was equipped with a multielectrode-cavity-array (MECA) chip (IONERA, Germany) to perform single-channel current recordings. The electrolytic buffer consists of 1 M KCl and 10 mM HEPES at pH 7.4. Before measurements, an appropriate volume of DPhPC solution (10 mg/mL in octane) should be smeared on the MECA chip to form a planar lipid bilayer. The successful formation of planar lipid bilayers can be verified by monitoring the resistance and capacitance of the corresponding cavities. Meanwhile, each cavity of the MECA chip was divided into two sides: cis side and trans side. Cholesterol-tagged DNA nanopores (1 nM) were then added to the cis side for spontaneous insertion into the planar lipid bilayer. The insertion process can be accelerated by adding a small volume of 0.5% n-octyloligooxyethylene (OPOE) to the cis side and monitored by increases in conductance steps. After successful insertion, triggers (10 nM) and reverse triggers (10 nM) were separately introduced to the cis side to induce the switching process of membrane-spanning DNA nanopore between the contracted state and expanded state, respectively. The constant voltage of + 80 mV was applied to monitor the dynamics of the embedded DNA nanopores. For molecular sensing experiments, an equal volume of trypsin solution (10 µM, PBS buffer) was added to the cis side of embedded DNP-C and DNP-E, respectively. Trypsin translocation events with different blocking amplitudes were recorded at + 50 mV. The Orbit Mini device was grounded at the trans side. Single-channel current traces were acquired at 10 kHz using Element Data Reader software (Elements, Italy). All obtained current traces were analyzed with Clampfit software (Molecular Devices, USA).

OxDNA simulations

Molecular dynamics simulations of DNP-E and DNP-C were performed using GPU-implemented sequence-dependent oxDNA2^41–43 in the canonical NVT (constant number of particles N, volume V, and temperature T) ensemble at 300 K and 1 M NaCl. Initial geometries of DNP-E and DNP-C were obtained using oxView’s rigid-body dynamics⁵². OxDNA simulations were performed using standard protocols as described in the previous study⁵² with the Anderson-like john thermostat. Both nanopores were energy minimized for 10,000 steps and relaxed for 30.3 𝜇s prior to production simulations that spanned 8 ms for DNP-C and DNP-E.

Ionic current calculations

From each of the simulated DNP-E and DNP-C oxDNA2 trajectories, we randomly selected ten configurations for the calculation of the ionic current, ensuring a minimum separation of at least 20 𝜇s between the chosen configurations. Subsequently, these chosen configurations were transformed into an all-atom representation via a custom script using the mrDNA package⁵³. Each of these all-atom configurations was embedded at the center of a DPhPC lipid bilayer patch (130 ×130 nm²) created by tiling a preequilibrated 13 ×13 nm² patch using VMD⁵⁴. Lipids having atoms within 15 Å of DNA atoms and those within the central cavity were both removed. Employing the volmap tool of VMD⁵⁴, we computed the three-dimensional map of the number density of lipid atoms with 1 Å resolution, marking voxels with a non-zero density as “occupied” by lipid. Equivalent maps were obtained for the DNA using the density of N1 purine atoms. For each occupancy map, the distance to the nearest occupied voxel was computed and used to look up the conductivity from a linear ramp for lipids, ranging from zero to the bulk conductivity value (11.18 S/m) between 3 and 10 Å, and a previously determined⁵⁵ tabulated dependence of the conductivity on distance from DNA. For each system, the two conductivity maps (lipid and DNA) were combined by using the minimum of the two values at each location. The resulting combined conductivity maps were used as inputs for the continuum SEM (steric exclusion model)⁴⁵, yielding simulated ionic currents. To validate the conductance obtained via SEM, we considered an analytical model of the conductance of a cylinder that fits within the hollow cavities of the DNP-C and DNP-E. The conductance of the cylinder is given by the bulk conductivity times the cylinder area over its height. In the case of the DNP-C, we positioned a cylinder with a radius of 9.5 nm and a height of 10 nm within the cavity, ensuring no overlap with the DNA. Correspondingly, a cylinder with a radius of 14.5 nm and a height of 10 nm was placed inside the DNP-E.

Confocal microscopy

Cholesterol-tagged DNP-C (10 nM) and DNP-E (10 nM) were incubated with GUVs solutions for 30 min, respectively, to ensure the spontaneous insertion of DNA nanopores into lipid membranes. These mixtures were then added separately to 35 mm µ-Dishes (Ibidi, Germany). Subsequently, each µ-Dish should be placed on the objective stage of the microscope and left for another 10 min to allow the GUVs to settle to the bottom of the corresponding µ-Dish. In a typical molecular influx assay, FITC-dextran (40 kDa, 2 µM) and TRITC-dextran (500 kDa, 2µM) were dropped together into the above µ-Dishes to investigate the size selectivity of DNP-C and DNP-E in controlling molecular transport. Next, a larger number of GUVs treated with DNP-C and DNP-E were dynamically monitored with a Nikon C2 confocal microscope (Nikon, Japan) at 20× or 40× magnification for 3 h. Fluorescent intensity changes of the obtained confocal images were measured and analyzed using ImageJ software (NIH, USA).

Statistical analysis

Current traces and images are shown as representative of all independent experiments. All data are presented as mean ± SD unless otherwise stated. Data analysis was performed using a GraphPad Prism 9.5 (GraphPad, USA). A two-tailed Student’s t-test was applied to evaluate the difference between the two groups. P-values less than 0.05 were considered statistically significant.

Acknowledgements

This research was supported in part by the National Natural Science Foundation of China under Grant 62273052, the Beijing Natural Science Foundation under Grant IS23062, and in part by the Grant-in-Aid for Scientific Research under Grant 22H01441 from the Ministry of Education, Culture, Sports, Science and Technology of Japan. H.C., C.M., and A.A. acknowledge support from the Human Frontier Science Project (RGP0047/2020), and also the supercomputer time provided through the ACCESS allocation grant (MCA05S028).

Author contributions

X.L. and F.L. conceived the idea of the project. T.A., Q.H., and X.L. supervised the project. F.L. constructed and characterized the DNA nanopores. F.L. and X.L. performed all electrophysiological experiments. H.C., C.M., and A.A. performed all computational characterizations. F.L. conducted the gel electrophoresis and confocal microscopy. F.L. and H.C. drew the schematic illustrations. F.L., X.L., and H.C. collected and interpreted data. X.L. and F.L. wrote the manuscript. All authors reviewed the manuscript.

Competing interests

The authors declare no competing interests.

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A dynamically gated triangular DNA nanopore for molecular sensing and cross-membrane transport

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Abstract

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Introduction

Results

Triangular DNA nanopore

Electrical characterization

Computational analysis

Protein sensing

Cross-membrane transport

Discussion

Methods

Materials

Preparation of DNA nanopore

Preparation of lipid vesicles

Agarose gel electrophoresis

TEM characterization

Single-channel current recordings

OxDNA simulations

Ionic current calculations

Confocal microscopy

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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