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Article Cite This: ACS Appl. Bio Mater. 2019, 2, 1278−1285
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One-Dimensional Assemblies of a DNA Tetrahedron: Manipulations on the Structural Conformation and Single-Molecule Behaviors Liying Wang,†,‡ Zhentong Zhu,§,∥ Bingling Li,*,§ and Fangwei Shao*,†,‡
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†
Zhejiang University−University of Illinois at Urbana-Champaign Institute, Zhejiang University, Haining 314400, People’s Republic of China ‡ Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 637371 § State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun, Jilin 130022, People’s Republic of China ∥ University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *
ABSTRACT: DNA nanotechnology can construct various nanostructures with diverse functionalities. However, conformation fluctuations due to the structural flexibility of duplex DNA compromise the efficiency to realize the functionality and reactivity of DNA nanostructures. To understand and control the structural deviation from the design represents a major challenge as well as an opportunity for DNA nanotechnology. In the present work, two series of one-dimensional assemblies of DNA tetrahedrons (DTHs) were fabricated and applied to demonstrate the manipulations of conformation dynamics of a one-dimensional DTH assembly by simple variation on linkage styles at singlemolecule resolution. A stepwise strategy allows both nanoassembly with a high fidelity in the number and sequence of DTH units to be assembled with a minimum number of linkage sequences. The characterization for these nanostructures with atomic force microscope (AFM) and a solid-state nanopore technique indicates the difference in conformation dynamics and bending stiffness between two analogous nanoassemblies both in the immobilized state on the surface and free state in solution. This work showed the power of fine-tuning the dynamic conformation of the nanostructures and could see the applications in singlemolecule biosensing and functionalization of DNA nanostructures. KEYWORDS: one dimension, DNA tetrahedron, conformation dynamics, single-molecule analysis, solid-state nanopore
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INTRODUCTION DNA nanotechnology has shown unprecedented power in constructing diverse nanostructures in one (1D) to three dimensions (3D).1,2 DNA origami, which folds long scaffold DNA into a target shape with the assistance of short staple strands, has become a promising method to fabricate complicate nanostructures.3,4 Alternatively, the tile-to-tile methods can extend 3D DNA nanostructures entirely with short synthetic oligonucleotides.5,6 Recently, with the emergence of much more diverse designs, the impact of DNA nanostructures has rapidly spread to a plethora of areas including nanofabrication,7,8 biomedicine,9−11 biosensing,12,13 and so on. However, the conformation dynamics, often observed as structural deviation between the design and the real status in solution or on the surface, especially in the case of high-order DNA nanoassemblies, can no longer be ignored, since the conformational discrepancy would significantly affect the functionality of the desired nanostructure.14,15 Though the structural flexibility is considered a major challenge in © 2019 American Chemical Society
achieving the aiming morphology of DNA nanostructure, it also represents a unique opportunity for biosensing and biomedical applications via manipulation of the conformation dynamics of DNA nanoassembly. Therefore, high demands have risen on understanding the relationship between the design and structural conformations of DNA nanostructure assembly. 1D assembly of DNA nanostructures is the simplest and most widely used DNA nanostructure in the field of nanomaterials, biomedicine, molecular device, and DNA computing.16 The morphology of 1D architectures, including sequence, length, shape, and bending stiffness, are important factors that will affect the properties and functionalities of 1D nanoassemblies.17−19 The majority of 1D DNA nanostructures obtained until now, either by DNA origami or the tile-to-tile Received: December 26, 2018 Accepted: January 29, 2019 Published: January 29, 2019 1278
DOI: 10.1021/acsabm.8b00834 ACS Appl. Bio Mater. 2019, 2, 1278−1285
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ACS Applied Bio Materials strategy, are as rigid as nanorods.20−22 However, recent studies suggested the flexibility of the 1D nanostructures could be essential to realizing the functionality, and there have been attempts to control the 1D bending of the DNA nanowire.15,23,24 Further manipulating the flexibility of the softwire type of DNA nanoassembly remains highly challenging, though it is necessary and useful for DNA nanostructures to have many functionalities in biosensing, molecular device, etc. The DNA tetrahedron (DTH), the simplest 3D nanostructure assembled by DNA oligonucleotides, has shown diverse applications in many fields.25,26 Besides the advantages like being highly achievable, stable, modifiable, and multifunctional,27−33 the capability of the DTH as a building block for higher-order nanoassembly was verified in recent publications.34−37 A 1D DTH trimer and a series of cyclic structures showed clearly the great potential to assembly of comprehensive nanostructures by DTHs in multiple dimensions.34−37 Both Wang and our group observed apparent deviation from linear conformation when DTH trimers are connected via a single linkage between the vertexes of adjacent DTHs. The linear assembly exceeding two DTHs cannot avoid a set of diverse ensembles of curve nanostructures, which is a result of both the inherent tetrahedral orientation of four vertexes and the thermal flexibility of the duplex linkages. However, if considering this from another side, this feature is beneficial to prepare the highly flexible softwire structure. Inspired by the cyclic dimer from our previous work,36 we prepared here a 1D softwire type of DTH with enhanced stiffness by using a cyclic dimer as the building blocks. Hence, two types of 1D DTH nanoassemblies with tunable length, size, and structural stiffness were obtained by using a stepwise assembly method. The characterization for these 1D nanostructures at the singlemolecule level with atomic force microscope (AFM) and solidstate nanopore electrophoresis indicate the conformation dynamics and bending stiffness of 1D DTH assemblies, even though as a softwire structure, they can be readily manipulated with simple alternation on the linkage style.
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Construction of LnD. In general, DTH dimers were formed by mixing two DTHs with equal molar quantity at 100 nM at room temperature overnight. The crude products were concentrated by centrifugal filtration and purified by SEC. The DTH assembly was constructed by stepwise mixing of two dimers followed with purification by SEC and concentration steps. Each step can achieve yield 70% or above. • L3D: D1′, 2D1, and 2′D were mixed with equal molar quantity and incubated overnight at room temperature. The crude product was concentrated by centrifugal filtration and purified by SEC. • L4D: Dimer (D1′ + 2D1) and dimer (D1′ + 2′D1′) with the same OD (1∼2 OD) were mixed and incubated overnight at room temperature. • L6D: Three dimers, D1′ + 2D1, 2′D1 + 3D1′, and 3′D1 + D1′ were assembled separately. Tetramer was formed by mixing the first two dimers. Then, tetramer and the last dimer were mixed together to form L6D. • L8D: Dimers I (D1′ + 2D1), II (2′D1 + 3D1′), and III (3′D1′ + 2′D1) were assembled. Dimers I and II were mixed to form a tetramer. Then, tetramer was mixed with dimer III to form a hexamer. Finally the one-dimensional extension was closed by mixing hexamer with dimer I to form L8D. Construction of LnS. LnS were procured with exactly the same procedure as LnD, except the S series of the DTHs listed in Table S2 was used for assembly. The composition of the S series of the DTHs are listed in Table S2. SEC Purification and Characterization. Sample (100 μL) was injected into the UFLC system and eluted with isocratic mobile phase (10 mM Tris-HCl, pH 7.3, 450 mM NaCl) with a flow rate of 0.5 mL/min. The target product peak was collected and concentrated by centrifugal filtration. AFM Characterization of Different Structures. Freshly cleaved mica was treated with 0.1% (v/v) APTES ((3-aminopropyl)triethoxysilane) aqueous solution before use.29 The SEC-purified DNA structure (40 μL, 2 nM) was dropped onto the mica surface and stood for 5 min. Then, the sample was scanned under the ScanAsyst in fluid mode with the ScanAsyst fluid + probe (Bruker, Singapore). Nanopore Translocation Experiment. The conical glass nanopores were fabricated by using a CO2-laser-actuated pipet puller (model P-2000, Sutter Instrument Co.) with a one-line program. After characterization under TEM, the nanopores were assembled into in house-made horizontal-type glass cells for the translocation experiment. The inner cavity of the glass nanopore and the cell acted as the trans and cis reservoirs, respectively. A chlorinated silver electrode was inserted into each reservoir. A potential of 200 mV was applied to the electrode inside the nanopore. The ion currents were collected with a current amplifier Axopatch 200B (Molecular Devices) using a lowpass Bassel filter of 10 kHz and digitized with a DigiData 1440A digitizer (Molecular Devices) at a sample rate of 100 kHz. The rootmean-square (rms) value of the measurement in this experiment is 1.1 pA. The DNA mixture (in 25 mM Tris-HCl, 640 mM NaCl, 5 mM KCl, and 0.5 mM EDTA, pH 7.5) was added into the cis reservoir. The current signal is processed using Clampfit 10.6 software (Molecular Devices).
EXPERIMENTAL SECTION
Materials. DNA oligonucleotides were purchased from Sangon (Shanghai, China), twice purified by HPLC. Chemicals for tris-Cl buffer and agarose gel were purchased from Bio-Rad (Singapore). DNA ladder (100bp) was purchased from Thermo Fisher (Singapore). Ultrapure water at 18.2 MΩ·cm was used in all experiments. Apparatus. The sample was concentrated by centrifugal filtration using Amicon Ultra, 0.5 mL Centrifugal Filters at 100 K. UV absorbance was measured on a Shimadzu UV-1800 spectrophotometer. A Shimadzu UFLC system with a BioSep-SEC-S 3000 column (Phenomenex, 300 × 7.8 mm) was used by monitoring the SEC chromatograph at 260 nm. Agarose gel was run on a horizontal electrophoresis system (Bio-Rad Wide Mini-Sub Cell GT Cell). AFM images were obtained on a Bruker Multimode 8 SPM equipped with a liquid cell. Construction of DNA Tetrahedron Building Blocks. The sequences of DNA oligonucleotides used are listed in Table S1. The stock solutions of four DNA oligonucleotides were prepared in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 450 mM NaCl), and the concentration was determined by UV absorbance at 260 nm. To anneal a DTH, four DNA oligonucleotides as listed in Table S2 were mixed with equal molar quantities in TE buffer to make a final concentration of 100 nM for each strand. The mixture solution was heated at 95 °C for 10 min and cooled to room temperature. The tetrahedrons in Table S2 were used directly without further purification in the assembly of LnS and LnD.
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RESULTS AND DISCUSSION Design and Fabrication of 1D Nanoassemblies of DTH. A single DNA tetrahedron was assembled with four single-stranded oligonucleotides via a thermal annealing method.25,36 To make the design as simple as possible, the sequences of the tetrahedral skeleton core were kept the same for all the DTHs used in this work (S1 to S4 in Table S1). Only the sequences of the linkages as single-stranded overhangs at the tetrahedral vertexes were varied accordingly. In order to construct 1D structures with different stiffness, DTHs were extended in a 1D manner via two linkage types by tuning the numbers and sequences of overhangs. In the first 1279
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Figure 1. Schematic illustration of 1D assembly design with DNA tetrahedrons as the building blocks. (A) The stepwise assembly of 1D nanostructures with a double linkage between adjacent tetrahedrons; detailed information about the sequences of the linkages is ignored for simplicity. (B) The detailed structure of the DTH octamer assembly with a single (L8S) or double (L8D) linkage; the linkages in blue, red, and black represent the sequences of 11′, 22′, and 33′, respectively.
Figure 2. Agarose gel electrophoresis (2.5%) (A) and SEC chromatogram (B) of LnS and LnD.
type, the adjacent DTHs are linked with each other by a single duplex, which is like a single-bond connection in n-alkane. Meanwhile, in the second type, two vertexes of each DTH are connected simultaneously to an adjacent DTH as dual linkages, similar to the double-bond connection in cumulene (Figure 1). In the second type, the free rotation of each single tetrahedron along the extension axis is confined by the doublelinkage design and is expected to enhance the bending stiffness.
Herein, the two 1D nanoassemblies are named as LnS or LnD accordingly (n represents the number of tetrahedron units, equal to 3, 4, 6, or 8; S or D annotates the nanoassembly as having a single or double linkage, respectively). Both LnS and LnD are assembled in a stepwise extension fashion as illustrated in Figure 1A. In general, in the initiation step, an initial dimer (D11′D2, 1, 1′, and 2 are the sequence numbers of the overhangs) was assembled by 2D1 and D1′ to 1280
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Figure 3. AFM characterization of LnS and LnD nanoassemblies. (A) AFM images of DTH nanoassemblies. (B) Histograms of end-to-end distance of DTH nanoassemblies in the AFM images. The red curves in B are the Gaussian fitting curves of the end-to-end distance. The mean value and standard deviation are indicated in each histogram.
the number of tetrahedron units (Figure 2A). The mobility of these structures perfectly matched the theoretical molecular size of each entity (Table S3) referring to the DNA ladder, whereas each pair of LnS and LnD with the same number of tetrahedron units showed little distinction in the mobility because the difference in bp numbers as a result of the variation in linkage style only accounts for less than 10% of total bp numbers. The structural morphology beyond those directly proportional to bp numbers, such as charges and molecular weights, are too subtle to be distinguished by the agarose gel electrophoresis. The DTH assemblies were further analyzed by size-exclusive chromatography (SEC), which is a separation method based upon the average globular size of bulk ensembles. As shown in Figure 2B, the retention times (TRs) for LnS with unit numbers of 3, 4, 6, and 8, are 12.84, 12.10, 11.37, and 11.03 min, respectively, while those for LnD are 13.21, 12.28, 11.46, and 11.13 min. Similar as that on gel electrophoresis, both families of DTH assemblies showed a shorter retention time for the 1D assembly with more DTH units, consistent with a larger molecular size in theory, whereas, all of the LnD showed a slightly longer retention time on the SEC column than that of the LnS analogues. The difference in retention time (ΔTR = TR(LnD) − TR(LnS)) declines with an increasing number of DTH, even though ΔTR for the L3 pair was already not large enough to separate L3S and L3D. However, the discrepancy in SEC retention between LnD and LnS analogue pairs, though very minimum, suggests that there could be a structural difference between LnS and LnD even after averaging over a large ensemble set. However, SEC still cannot allow us to identify the difference in the structural morphology or even
form a double linkage (11′) within the dimer and two singlestranded overhangs (2) at two vertexes of the second DTH on the desired 1D assembly. In the following chain elongation process, each step introduced a dimer (mD11′Dn, m and n are the sequence numbers of double overhangs on each DTH) to the chain as a building block unit. Finally, the 1D extension was terminated by either a single DTH or a dimer for the DTH assembly with an odd or even number of DTHs, respectively. To achieve maximum simplification in linkage design, all the dimers used the same internal double-linkage sequence, 11′. Only two pairs of linkage sequences, 22′ and 33′, are used in alternative steps for the dimer extension. Because of the fact that only one pair of linkages is exposed as a single-stranded overhang in each step, in theory, only these two pairs of duplex sequences are necessary to accomplish 1D extension with an unlimited number of DTHs, though a maximum eight DTH units in one assembly are demonstrated in this work. In total, only seven DTHs are used to assemble all LnD (Table S2). This stepwise extension strategy allows only two counterparts to participate in each isothermal-annealing step, which would ensure the hybridization efficiency. By removing one overhang from each DTH, LnS were assembled with the same step strategy. The detailed structures of each LnS and LnD are illustrated in Figure 1B and Figure S1. The preparation process was described in detail in the Experimental Section. Bulk Ensemble Characterization by Agarose Gel Electrophoresis and SEC. The successful assembly of two series of DTH nanoassemblies were verified, and the structural morphology were first characterized by agarose gel electrophoresis. The extension of the 1D nanoassembly was clearly shown by the decline in band migration along with increasing 1281
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Figure 4. Solid-state nanopore electrophoresis of L6S and L6D by a conical glass nanopore (CGN). (A) Illustration of L6D nanoassembly traversing CGN. (B) Typical current trace of L6D (top) and L6S (bottom) vs time extracted from a 1−2 h recording. (C,D) Scatter plots of current drop vs duration of DNA translocation for pure L6S (C, purple), L6D (C, orange), and a 1:1 mixture of the two nanoassemblies (D, green). (E,F) All-point current drop histograms of pure L6S (E, purple), L6D (E, orange), and the mixture (F, green). (G) A typical current trace of L6D and L6S mixture vs time extracted from a 1−2 h recording.
counted for each sample, and the distance distribution is shown in Figure 3B. If comparing the histogram of LnS and LnD with the same unit number, the average distances are similar for n = 3 or 4. For short assemblies, the linear structures still could be reached by both types of assemblies, or the backbone curling is not severe yet. However, the increment of distance from L3D to L4D (11.2 nm) is larger than that of LnS (7.6 nm), indicating that the rigid effect from the double linkage started to emerge even in the short assemblies. The difference in the average lengths is more pronounced when n reaches 6 and 8. L6D showed an ∼12 nm increment from L4D, when only a 4 nm increment was obtained for L6S. More remarkably, the distance discrepancy between L8D and L8S soared to 21.6 nm, while only a 4 nm extension was obtained from L6S to L8S. The average end-to-end distance of the LnD series escalated linearly with >11 nm elongation for every addition of a dimer unit. A near linear increment of the end-toend distance suggested that the LnD assemblies could maintain a quite high bending stiffness and behave much closer to a linear chain or rod as expected by design, while LnS series deviate ominously from the linear conformation. However, unlike 1D wireframe DNA origami, LnD assemblies still possess backbone flexibility to a certain degree. For L8D, two main peaks can be fitted to the distribution histogram. One peak was centered at 47.1 ± 6.1 nm, representing the bended structures; the other peak at 71.8 ± 16.4 nm was the population of slightly bended or linear structures. For such a long LnD assembly, the DTH chain can bend at a certain linkage position, while the rest of chain holds linear conformation as two or several short rods. Hence, AFM measurement provides a snapshot of the conformation dynamics of both DTH assemblies upon immobilizing individual structures on the surface. Both images and statistical analysis indicate that the dynamic conformation and bending stiffness of the DTH assembly are varied according to the
just isolate the two types of 1D nanoassembly, especially when the DTH number increases, and the size of 1D nanoassembly approaches the separation limitation of SEC column. Single-Molecule Characterization by AFM and SolidState Nanopore Technology. To elucidate the structural morphology between LnS and LnD, a series of measurements at the single-molecule level were carried out for both 1D nanoassemblies. First, the structural conformation of the assemblies was characterized by fluid mode AFM (Figure 3A). DTH assemblies were instantly immobilized on the mica surface by electrostatic attraction, and the conformation of individual nanoassembly molecules could be locked on a 2D surface and be visualized with nanometer resolution. The conformations of LnS molecules are apparently the softwire type. Even as short as L3S, very few linear conformations were observed in AFM image, while the majority of assembly molecules bent at the central DTH and showed a variety of curvatures. With the addition of DTH units, no linear conformation could be visualized from L4S up to L8S. LnS ensembles twisted severely with larger unit number. Random coiling conformations as a condensed cluster of DTHs emerged from L4S and became the major conformation in the AFM image of L8S. On the contrary, as shown in the lower panel of Figure 3A, many more LnD ensembles can retain their linear conformation. The majority of L3D and L4D molecules showed a short rod structure. Even in the L8D image, the linear conformation could be spotted. Many ensembles only bent at one or a couple of the DTH sites and showed an extended or “open” conformation compared to the cluster conformation of L8S. Hence, the backbone flexibility along the assembly axis is significantly reduced, and bending stiffness is enhanced in comparison to LnS structures. The bending stiffness of LnS and LnD was further analyzed statistically by measuring the end-to-end distance of individual ensembles in AFM images. At least 200 structures were 1282
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conformation dynamics resulting from the variation of linkage patterns in the DTH nanoassembly can be readily observed by nanopore electrophoresis, for either pure or mixture samples.
linkage pattern, while other properties of the assembly such as number and sequence of the DTH unit remain the same. To study the conformation dynamics of DTH nanoassemblies in solution phase, we used solid-state nanopore electrophoresis. Nanopore electrophoresis is a promising single-molecule analysis technology that can detect a wide range of species such as DNA, proteins, and small molecules.38−49 The detection principle lies in the transient current drop when an inert polymer is driven by voltage to pass through a nanopore.50 With the advantages of high-throughput capacity, size-exclusive recognition, and a completely homogeneous working condition, this technique has successfully detected a variety of nucleic acid nanostructures, such as the hybridization chain reaction (HCR) products,51 DNA cubes/ RNA rings,52 and linear/branched dsDNA.53 These recent work suggests that the nanopore technique could have the potential to distinguish a subtle conformation difference of DNA nanostructures. Here, we used a conical glass nanopore (CGN) to examine both types of nanoassemblies, since the diameter of nanopore (∼12 nm) is suitable to allow the translocation of the DTH (theoretical edge length ≈ 6.8 nm) used in our work (Figure S3). The pore size is less than twice of the spherical diameter of a DTH, which is also the nominal width of DTH 1D assemblies in the linear conformation. Four samples, L3S, L3D, L6S, and L6D, were analyzed under the same working condition. As shown in Figure 4 and Figure S4, when the DTH assembly was electrophoretically driven through the nanopore, both DTH hexamers showed a larger current drop and longer duration time than those of the DTH trimer. Both electric parameters can distinguish DTH assemblies with a substantial length difference that was due to the composition variation, such as different unit numbers of the building block. Further analysis of the electrophoretic properties between the assemblies with the same unit number revealed more subtle conformation dynamics in the solution phase. L3S and L3D showed similar current blockage, which suggests that both DTH trimers occupy the similar inner space during the translocation process (Figure S4). Meanwhile, for DTH hexamers, a significant difference in the current blockage was observed. L6S showed a current drop of 77.8 ± 7.2 pA, which was 2.6 times greater than that of L6D (29.5 ± 4.9 pA). This is consistent with the conformational features of the two structures revealed by AFM (Figure 3A). With double linkages, L6D showed a much higher possibility of maintaining a linear conformation as a nanometer molecular rod. When L6D was translocated through the nanopore, the extended conformation induced a small current drop around 30 pA, whereas the large current drop for L6S ensembles indicated L6S, because of the flexibility of the single-linkage design, was nearly dominated by the curling conformation as shown in the AFM image. Even though electrophoretic force may stretch L6S and induce a slightly open conformation, a majority of L6S molecules may still adopt a curled nonlinear conformation when they were passing through the glass nanopore. The significant difference in current blockage even allows an easy separation of the two nanoassemblies when L6S and L6D were submitted to nanopore analysis simultaneously in a mixture sample solution (Figure 4D,F,G). Two distinctive current drop peaks emerged at a similar pA as isolated L6D and L6S ensembles, respectively, which suggests no interference on the conformation dynamics occurs between two types of hexamers during the nanopore translocation process. The difference in
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DISCUSSION Two series of DTH nanoassemblies were fabricated by a stepwise isothermal-annealing protocol. There are several advantages in this assembly design. First, the stepwise assembly provides a procedure to allow stepwise extension of the DTH assembly in one dimension. With only two counterparts in each annealing step, this protocol ensures that the linear structures can be achieved with a precise number and defined sequence of DTHs, while only three sequences of duplex linkages are required. One sequence for an internal linkage of dimers and two used alternatively to link the incoming dimers is the simplest linkage sequence design we used in the present work. Second, the assembly design readily adopts variation on the tetrahedron cores. By introducing a DTH dimer or monomer with distinctive edge sequences, a sequence of DTHs can encoded into the 1D nanoassembly. Precise modification and functionalization on the edge, vertex, and linkage of the DTH can provide several layers of coding framework, which can be read as whole or separate systems. DTH assembly can provide templates for various subjects on the nanometer scale, such as inorganic nanoparticles, optical probes, molecular walkers, proteins, and antibodies, among others. Third, the assembly protocol can be extrapolated to assemble DTH dimers in two and three dimensions by introducing dual overhangs at the vertex. From one initial DTH monomer or dimer, two lines of DTH extension could progress in parallel to achieve dendritic or other complicate DTH assembled nanostructures. To manipulate the conformation dynamics of DTH nanoassemblies, we make LnS and LnD maintain the maximum similarity in the composition. Each pair of analoguess of LnS and LnD contains the same number and edge sequence of DTH units as well as the same sequence and order of duplex linkages and only differ at the number of duplex linkages between the adjacent DTHs. These two designs of 1D assembly would allow all the differences in conformation dynamics and bending stiffness to be attributed to the assembly design. Tetrahedron units are analogous to carbon atoms, and single duplex linkages are analogous to single sigma bonds. Each individual DTH in an LnS assembly can rotate freely around the linked vertex without cooperatively dragging the adjacent DTH. LnS assemblies are analogous to n-alkanes with high backbone flexibility and with little bending stiffness. On the other hand, with a second linkage between one more vertex pairs of adjacent DTHs, the connection pattern in LnD is analogous to cumulene with continuous double bonds. Instead of a softwire structure, LnD assemblies showed a worm-like shape with bending of 1D assembly only occurring to one or two linkage sites. DTHs in LnD are connected at two vertexes. The pair of double linkages is locked into one plane. The adjacent linkage planes are perpendicular to each other. The rotation of DTH restrains the adjacent double-linkage planes to 2D 360° around the DTH edge, like a door hinge. The subtle variation only in the linkage patterns results in high similarity in globular size of the DTH nanostructures. The two families of DTH assemblies showed little difference in bulk ensemble measurements, such as agarose gel electrophoresis and size-exclusive chromatography. Only with single-molecule measurements, the conformation 1283
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dynamics of individual assembly and the effects from linkage style on the conformation rigidity and bending stiffness of 1D assembly were revealed. LnS showed much lower bending stiffness compared to LnD. AFM showed that with the 1D assembly extended, fewer assemblies of LnS could stretch out in an “open” conformation, not even the mentioned linear rod shape. Whereas, unlike LnS, LnD no longer falls into the softwire style but behaves more like a worm, which curls only at a few joints and allows the entire assembly to move as several short rods connected together. The difference in conformation dynamics results in the large discrepancy of current blockage during the electrophoretic translocation of the DTH assembly through glass nanopore. The fact that large DTH assemblies with subtle differences in composition can be readily distinguished by nanopore electrophoresis even in a mixture solution could be seen as a promising method for single-molecule biosensing based upon the stimulus-responsive conformation alteration of DTH nanoassembly. A simple alteration on linkage number can manipulate the bending stiffness and consequential conformation dynamics of the highordered nanostructure without removing or scrabbling the inherent sequence of nanobuilding blocks. This approach would endow a wide variety of reactive and computing functionalities to the DNA nanostructure as a molecular device, logic gate, and structure template.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected] (F.S.) *E-mail: [email protected] (B.L.) ORCID
Fangwei Shao: 0000-0003-2007-3920 Funding
This work is supported by a Singapore AcRF MOE Tier 1 grant (M4011554), the K.C. Wong Education Foundation, China, and a start-up grant from Zhejing University. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Zhang, F.; Nangreave, J.; Liu, Y.; Yan, H. Structural DNA Nanotechnology: State of the Art and Future Perspective. J. Am. Chem. Soc. 2014, 136, 11198−11211. (2) Seeman, N. C.; Sleiman, H. F. DNA nanotechnology. Nat. Rev. Mater. 2017, 3, 17068. (3) Rothemund, P. W. K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297−302. (4) Douglas, S. M.; Dietz, H.; Liedl, T.; Högberg, B.; Graf, F.; Shih, W. M. Self-assembly of DNA into Nanoscale Three-dimensional Shapes. Nature 2009, 459, 414−418. (5) Winfree, E.; Liu, F. R.; Wenzler, L. A.; Seeman, N. C. Design and Self-assembly of Two-dimensional DNA Crystals. Nature 1998, 394, 539−544. (6) He, Y.; Ye, T.; Su, M.; Zhang, C.; Ribbe, A. E.; Jiang, W.; Mao, C. Hierarchical Self-assembly of DNA into Symmetric Supramolecular Polyhedra. Nature 2008, 452, 198−201. (7) Yan, H; Park, S. H.; Finkelstein, G.; Reif, J. H.; LaBean, T. H. DNA-Templated Self-Assembly of Protein Arrays and Highly Conductive Nanowires. Science 2003, 301, 1882−1884. (8) Aldaye, F. A.; Palmer, A. L.; Sleiman, H. F. Assembling Materials with DNA as the Guide. Science 2008, 321, 1795−1799. (9) Chen, Y. J.; Groves, B.; Muscat, R. A.; Seelig, G. DNA Nanotechnology from the Test Tube to the Cell. Nat. Nanotechnol. 2015, 10, 748−760. (10) Kumar, V.; Palazzolo, S.; Bayda, S.; Corona, G.; Toffoli, G.; Rizzolio, F. DNA Nanotechnology for Cancer Therapy. Theranostics 2016, 6, 710−725. (11) Pei, H.; Zuo, X.; Zhu, D.; Huang, Q.; Fan, C. Functional DNA Nanostructures for Theranostic Applications. Acc. Chem. Res. 2014, 47, 550−559. (12) Chao, J.; Zhu, D.; Zhang, Y.; Wang, L.; Fan, C. DNA Nanotechnology-enabled Biosensors. Biosens. Bioelectron. 2016, 76, 68−79. (13) Ye, D.; Zuo, X.; Fan, C. DNA Nanotechnology-Enabled Interfacial Engineering for Biosensor Development. Annu. Rev. Anal. Chem. 2018, 11, 171−195. (14) Kim, D.-N.; Kilchherr, F.; Dietz, H.; Bathe, M. Quantitative Prediction of 3D Solution Shape and Flexibility of Nucleic Acid Nanostructures. Nucleic Acids Res. 2012, 40, 2862−2868. (15) Benson, E.; Mohammed, A.; Rayneau-Kirkhope, D.; Gadin, A.; Orponen, P.; Hogberg, B. Effects of Design Choices on the Stiffness of Wireframe DNA Origami Structures. ACS Nano 2018, 12, 9291− 9299. (16) Wang, F.; Willner, B.; Willner, I. DNA Nanotechnology With One-dimensional Self-Assembled Nanostructures. Curr. Opin. Biotechnol. 2013, 24, 562−574. (17) Geng, Y.; Dalhaimer, P.; Cai, S.; Tsai, R.; Tewari, M.; Minko, T.; Discher, D. E. Shape Effects of Filaments versus Spherical Particles in Flow and Drug Delivery. Nat. Nanotechnol. 2007, 2, 249−255. (18) Yuan, B.; Cademartiri, L. Flexible One-Dimensional Nanostructures: A Review. J. Mater. Sci. Technol. 2015, 31, 607−615.
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CONCLUSION In conclusion, we have designed and fabricated two series of one-dimensional nanostructures with a defined number and sequence of DNA tetrahedrons. With simple variation in the number of linkages, the conformation dynamics of DTH nanostructures can be well controlled. The characterization with AFM and the solid-state nanopore technique revealed the conformational structures of LnS and LnD. Both techniques showed higher linear rigidity of LnD compared to LnS, especially for large assemblies. This is the first time that it is shown that the 1D bending stiffness of DNA nanostructures is dynamic both on the surface and in solution at single-molecule level, which may pave the way to understanding the dynamic behavior of DNA nanostructures as molecular devices and biomedical nanovessels. In addition, the work also demonstrated the power of the solid-state nanopore technique in analyzing the dynamic conformation of nanostructures and in sensing subtle conformation changes in biomolecular nanostructures. Profound studies in the future on many important factors like pore size and driving voltage may provide more definite correlation between the electrophoretic signals and nanostructures, which might open up a new era for the solidstate nanopore technique in the characterization and detection of biomacromolecules as well as tracking the reactions/ motions in nanodevices.
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Article
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00834. Oligonucleotide sequences for DNA tetrahedrons, additional structural information, plots, solid-state nanopore analysis (PDF) 1284
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(19) Wang, Z.; Wu, Z.; Liu, J.; Zhang, W. Particle Morphology: an Important Factor Affecting Drug Delivery by Nanocarriers into Solid Tumors. Expert Opin. Drug Delivery 2018, 15, 379−395. (20) Douglas, S. M.; Chou, J. J.; Shih, W. M. DNA-nanotubeinduced Alignment of Membrane Proteins for NMR Structure Determination. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 6644−6648. (21) Aldaye, F. A.; Lo, P. K.; Karam, P.; McLaughlin, C. K.; Cosa, G.; Sleiman, H. F. Modular Construction of DNA Nanotubes of Tunable Geometry and Single- or Double-stranded Character. Nat. Nanotechnol. 2009, 4, 349−352. (22) Kuzyk, A.; Schreiber, R.; Fan, Z.; Pardatscher, G.; Roller, E.-M.; Högele, A.; Simmel, F. C.; Govorov, A. O.; Liedl, T. DNA-based Selfassembly of Chiral Plasmonic Nanostructures with Tailored Optical Response. Nature 2012, 483, 311−314. (23) Zhou, L.; Marras, A. E.; Su, H.-J.; Castro, C. E. DNA Origami Compliant Nanostructures with Tunable Mechanical Properties. ACS Nano 2014, 8, 27−34. (24) Rahbani, J. F.; Hariri, A. A.; Cosa, G.; Sleiman, H. F. Dynamic DNA Nanotubes: Reversible Switching between Single and DoubleStranded Forms, and Effect of Base Deletions. ACS Nano 2015, 9, 11898−11908. (25) Goodman, R. P.; Berry, R. M.; Turberfield, A. J. The Single-step Synthesis of a DNA Tetrahedron. Chem. Commun. 2004, 1372−1373. (26) Goodman, R. P.; Schaap, I. A. T.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J. Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication. Science 2005, 310, 1661−1665. (27) Mastroianni, A. J.; Claridge, S. A.; Alivisatos, A. P. Pyramidal and Chiral Groupings of Gold Nanocrystals Assembled Using DNA Scaffolds. J. Am. Chem. Soc. 2009, 131, 8455−8459. (28) Walsh, A. S.; Yin, H. F.; Erben, C. M.; Wood, M. J. A.; Turberfield, A. J. DNA Cage Delivery to Mammalian Cells. ACS Nano 2011, 5, 5427−5432. (29) Kim, K. R.; Kim, D. R.; Lee, T.; Yhee, J. Y.; Kim, B. S.; Kwon, I. C.; Ahn, D. R. Drug Delivery by a Self-assembled DNA Tetrahedron for Overcoming Drug Resistance in Breast Cancer cells. Chem. Commun. 2013, 49, 2010−2012. (30) Wilks, T. R.; Bath, J.; de Vries, J. W.; Raymond, J. E.; Herrmann, A.; Turberfield, A. J.; O’Reilly, R. K. ″Giant Surfactants″ Created by the Fast and Efficient Functionalization of a DNA Tetrahedron with a Temperature-Responsive Polymer. ACS Nano 2013, 7, 8561−8572. (31) Mitchell, N.; Schlapak, R.; Kastner, M.; Armitage, D.; Chrzanowski, W.; Riener, J.; Hinterdorfer, P.; Ebner, A.; Howorka, S. A DNA Nanostructure for the Functional Assembly of Chemical Groups with Tunable Stoichiometry and Defined Nanoscale Geometry. Angew. Chem., Int. Ed. 2009, 48, 525−527. (32) Pei, H.; Lu, N.; Wen, Y. L.; Song, S. P.; Liu, Y.; Yan, H.; Fan, C. H. A DNA Nanostructure-based Biomolecular Probe Carrier Platform for Electrochemical Biosensing. Adv. Mater. 2010, 22, 4754−4758. (33) Zhu, D.; Pei, H.; Yao, G.; Wang, L.; Su, S.; Chao, J.; Wang, L.; Aldalbahi, A.; Song, S.; Shi, J.; Hu, J.; Fan, C.; Zuo, X. A SurfaceConfined Proton-Driven DNA Pump Using a Dynamic 3D DNA Scaffold. Adv. Mater. 2016, 28, 6860−6865. (34) Xing, S.; Jiang, D. W.; Li, F.; Li, J.; Li, Q.; Huang, Q.; Guo, L. J.; Xia, J. Y.; Shi, J. Y.; Fan, C. H.; Zhang, L.; Wang, L. H. Constructing Higher-Order DNA Nanoarchitectures with Highly Purified DNA Nanocages. ACS Appl. Mater. Interfaces 2015, 7, 13174−13179. (35) Barth, A.; Kobbe, D.; Focke, M. DNA-DNA Kissing Complexes as a New Tool for the Assembly of DNA Nanostructures. Nucleic Acids Res. 2016, 44, 1502−1513. (36) Wang, L.; Meng, Z.; Martina, F.; Shao, H.; Shao, F. Fabrication of Circular Assemblies with DNA Tetrahedrons: From Static Structures to a Dynamic Rotary Motor. Nucleic Acids Res. 2017, 45, 12090−12099. (37) Liu, Q.; Ge, Z. L.; Mao, X. H.; Zhou, G. B.; Zuo, X. L.; Shen, J. W.; Shi, J. Y.; Li, J.; Wang, L. H.; Chen, X. Q.; Fan, C. H. Valency1285
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One-Dimensional Assemblies of a DNA Tetrahedron: Manipulations on the Structural Conformation and Single-Molecule Behaviors Liying Wang,†,‡ Zhentong Zhu,§,∥ Bingling Li,*,§ and Fangwei Shao*,†,‡
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†
Zhejiang University−University of Illinois at Urbana-Champaign Institute, Zhejiang University, Haining 314400, People’s Republic of China ‡ Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 637371 § State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, Changchun, Jilin 130022, People’s Republic of China ∥ University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *
ABSTRACT: DNA nanotechnology can construct various nanostructures with diverse functionalities. However, conformation fluctuations due to the structural flexibility of duplex DNA compromise the efficiency to realize the functionality and reactivity of DNA nanostructures. To understand and control the structural deviation from the design represents a major challenge as well as an opportunity for DNA nanotechnology. In the present work, two series of one-dimensional assemblies of DNA tetrahedrons (DTHs) were fabricated and applied to demonstrate the manipulations of conformation dynamics of a one-dimensional DTH assembly by simple variation on linkage styles at singlemolecule resolution. A stepwise strategy allows both nanoassembly with a high fidelity in the number and sequence of DTH units to be assembled with a minimum number of linkage sequences. The characterization for these nanostructures with atomic force microscope (AFM) and a solid-state nanopore technique indicates the difference in conformation dynamics and bending stiffness between two analogous nanoassemblies both in the immobilized state on the surface and free state in solution. This work showed the power of fine-tuning the dynamic conformation of the nanostructures and could see the applications in singlemolecule biosensing and functionalization of DNA nanostructures. KEYWORDS: one dimension, DNA tetrahedron, conformation dynamics, single-molecule analysis, solid-state nanopore
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INTRODUCTION DNA nanotechnology has shown unprecedented power in constructing diverse nanostructures in one (1D) to three dimensions (3D).1,2 DNA origami, which folds long scaffold DNA into a target shape with the assistance of short staple strands, has become a promising method to fabricate complicate nanostructures.3,4 Alternatively, the tile-to-tile methods can extend 3D DNA nanostructures entirely with short synthetic oligonucleotides.5,6 Recently, with the emergence of much more diverse designs, the impact of DNA nanostructures has rapidly spread to a plethora of areas including nanofabrication,7,8 biomedicine,9−11 biosensing,12,13 and so on. However, the conformation dynamics, often observed as structural deviation between the design and the real status in solution or on the surface, especially in the case of high-order DNA nanoassemblies, can no longer be ignored, since the conformational discrepancy would significantly affect the functionality of the desired nanostructure.14,15 Though the structural flexibility is considered a major challenge in © 2019 American Chemical Society
achieving the aiming morphology of DNA nanostructure, it also represents a unique opportunity for biosensing and biomedical applications via manipulation of the conformation dynamics of DNA nanoassembly. Therefore, high demands have risen on understanding the relationship between the design and structural conformations of DNA nanostructure assembly. 1D assembly of DNA nanostructures is the simplest and most widely used DNA nanostructure in the field of nanomaterials, biomedicine, molecular device, and DNA computing.16 The morphology of 1D architectures, including sequence, length, shape, and bending stiffness, are important factors that will affect the properties and functionalities of 1D nanoassemblies.17−19 The majority of 1D DNA nanostructures obtained until now, either by DNA origami or the tile-to-tile Received: December 26, 2018 Accepted: January 29, 2019 Published: January 29, 2019 1278
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ACS Applied Bio Materials strategy, are as rigid as nanorods.20−22 However, recent studies suggested the flexibility of the 1D nanostructures could be essential to realizing the functionality, and there have been attempts to control the 1D bending of the DNA nanowire.15,23,24 Further manipulating the flexibility of the softwire type of DNA nanoassembly remains highly challenging, though it is necessary and useful for DNA nanostructures to have many functionalities in biosensing, molecular device, etc. The DNA tetrahedron (DTH), the simplest 3D nanostructure assembled by DNA oligonucleotides, has shown diverse applications in many fields.25,26 Besides the advantages like being highly achievable, stable, modifiable, and multifunctional,27−33 the capability of the DTH as a building block for higher-order nanoassembly was verified in recent publications.34−37 A 1D DTH trimer and a series of cyclic structures showed clearly the great potential to assembly of comprehensive nanostructures by DTHs in multiple dimensions.34−37 Both Wang and our group observed apparent deviation from linear conformation when DTH trimers are connected via a single linkage between the vertexes of adjacent DTHs. The linear assembly exceeding two DTHs cannot avoid a set of diverse ensembles of curve nanostructures, which is a result of both the inherent tetrahedral orientation of four vertexes and the thermal flexibility of the duplex linkages. However, if considering this from another side, this feature is beneficial to prepare the highly flexible softwire structure. Inspired by the cyclic dimer from our previous work,36 we prepared here a 1D softwire type of DTH with enhanced stiffness by using a cyclic dimer as the building blocks. Hence, two types of 1D DTH nanoassemblies with tunable length, size, and structural stiffness were obtained by using a stepwise assembly method. The characterization for these 1D nanostructures at the singlemolecule level with atomic force microscope (AFM) and solidstate nanopore electrophoresis indicate the conformation dynamics and bending stiffness of 1D DTH assemblies, even though as a softwire structure, they can be readily manipulated with simple alternation on the linkage style.
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Construction of LnD. In general, DTH dimers were formed by mixing two DTHs with equal molar quantity at 100 nM at room temperature overnight. The crude products were concentrated by centrifugal filtration and purified by SEC. The DTH assembly was constructed by stepwise mixing of two dimers followed with purification by SEC and concentration steps. Each step can achieve yield 70% or above. • L3D: D1′, 2D1, and 2′D were mixed with equal molar quantity and incubated overnight at room temperature. The crude product was concentrated by centrifugal filtration and purified by SEC. • L4D: Dimer (D1′ + 2D1) and dimer (D1′ + 2′D1′) with the same OD (1∼2 OD) were mixed and incubated overnight at room temperature. • L6D: Three dimers, D1′ + 2D1, 2′D1 + 3D1′, and 3′D1 + D1′ were assembled separately. Tetramer was formed by mixing the first two dimers. Then, tetramer and the last dimer were mixed together to form L6D. • L8D: Dimers I (D1′ + 2D1), II (2′D1 + 3D1′), and III (3′D1′ + 2′D1) were assembled. Dimers I and II were mixed to form a tetramer. Then, tetramer was mixed with dimer III to form a hexamer. Finally the one-dimensional extension was closed by mixing hexamer with dimer I to form L8D. Construction of LnS. LnS were procured with exactly the same procedure as LnD, except the S series of the DTHs listed in Table S2 was used for assembly. The composition of the S series of the DTHs are listed in Table S2. SEC Purification and Characterization. Sample (100 μL) was injected into the UFLC system and eluted with isocratic mobile phase (10 mM Tris-HCl, pH 7.3, 450 mM NaCl) with a flow rate of 0.5 mL/min. The target product peak was collected and concentrated by centrifugal filtration. AFM Characterization of Different Structures. Freshly cleaved mica was treated with 0.1% (v/v) APTES ((3-aminopropyl)triethoxysilane) aqueous solution before use.29 The SEC-purified DNA structure (40 μL, 2 nM) was dropped onto the mica surface and stood for 5 min. Then, the sample was scanned under the ScanAsyst in fluid mode with the ScanAsyst fluid + probe (Bruker, Singapore). Nanopore Translocation Experiment. The conical glass nanopores were fabricated by using a CO2-laser-actuated pipet puller (model P-2000, Sutter Instrument Co.) with a one-line program. After characterization under TEM, the nanopores were assembled into in house-made horizontal-type glass cells for the translocation experiment. The inner cavity of the glass nanopore and the cell acted as the trans and cis reservoirs, respectively. A chlorinated silver electrode was inserted into each reservoir. A potential of 200 mV was applied to the electrode inside the nanopore. The ion currents were collected with a current amplifier Axopatch 200B (Molecular Devices) using a lowpass Bassel filter of 10 kHz and digitized with a DigiData 1440A digitizer (Molecular Devices) at a sample rate of 100 kHz. The rootmean-square (rms) value of the measurement in this experiment is 1.1 pA. The DNA mixture (in 25 mM Tris-HCl, 640 mM NaCl, 5 mM KCl, and 0.5 mM EDTA, pH 7.5) was added into the cis reservoir. The current signal is processed using Clampfit 10.6 software (Molecular Devices).
EXPERIMENTAL SECTION
Materials. DNA oligonucleotides were purchased from Sangon (Shanghai, China), twice purified by HPLC. Chemicals for tris-Cl buffer and agarose gel were purchased from Bio-Rad (Singapore). DNA ladder (100bp) was purchased from Thermo Fisher (Singapore). Ultrapure water at 18.2 MΩ·cm was used in all experiments. Apparatus. The sample was concentrated by centrifugal filtration using Amicon Ultra, 0.5 mL Centrifugal Filters at 100 K. UV absorbance was measured on a Shimadzu UV-1800 spectrophotometer. A Shimadzu UFLC system with a BioSep-SEC-S 3000 column (Phenomenex, 300 × 7.8 mm) was used by monitoring the SEC chromatograph at 260 nm. Agarose gel was run on a horizontal electrophoresis system (Bio-Rad Wide Mini-Sub Cell GT Cell). AFM images were obtained on a Bruker Multimode 8 SPM equipped with a liquid cell. Construction of DNA Tetrahedron Building Blocks. The sequences of DNA oligonucleotides used are listed in Table S1. The stock solutions of four DNA oligonucleotides were prepared in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 450 mM NaCl), and the concentration was determined by UV absorbance at 260 nm. To anneal a DTH, four DNA oligonucleotides as listed in Table S2 were mixed with equal molar quantities in TE buffer to make a final concentration of 100 nM for each strand. The mixture solution was heated at 95 °C for 10 min and cooled to room temperature. The tetrahedrons in Table S2 were used directly without further purification in the assembly of LnS and LnD.
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RESULTS AND DISCUSSION Design and Fabrication of 1D Nanoassemblies of DTH. A single DNA tetrahedron was assembled with four single-stranded oligonucleotides via a thermal annealing method.25,36 To make the design as simple as possible, the sequences of the tetrahedral skeleton core were kept the same for all the DTHs used in this work (S1 to S4 in Table S1). Only the sequences of the linkages as single-stranded overhangs at the tetrahedral vertexes were varied accordingly. In order to construct 1D structures with different stiffness, DTHs were extended in a 1D manner via two linkage types by tuning the numbers and sequences of overhangs. In the first 1279
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Figure 1. Schematic illustration of 1D assembly design with DNA tetrahedrons as the building blocks. (A) The stepwise assembly of 1D nanostructures with a double linkage between adjacent tetrahedrons; detailed information about the sequences of the linkages is ignored for simplicity. (B) The detailed structure of the DTH octamer assembly with a single (L8S) or double (L8D) linkage; the linkages in blue, red, and black represent the sequences of 11′, 22′, and 33′, respectively.
Figure 2. Agarose gel electrophoresis (2.5%) (A) and SEC chromatogram (B) of LnS and LnD.
type, the adjacent DTHs are linked with each other by a single duplex, which is like a single-bond connection in n-alkane. Meanwhile, in the second type, two vertexes of each DTH are connected simultaneously to an adjacent DTH as dual linkages, similar to the double-bond connection in cumulene (Figure 1). In the second type, the free rotation of each single tetrahedron along the extension axis is confined by the doublelinkage design and is expected to enhance the bending stiffness.
Herein, the two 1D nanoassemblies are named as LnS or LnD accordingly (n represents the number of tetrahedron units, equal to 3, 4, 6, or 8; S or D annotates the nanoassembly as having a single or double linkage, respectively). Both LnS and LnD are assembled in a stepwise extension fashion as illustrated in Figure 1A. In general, in the initiation step, an initial dimer (D11′D2, 1, 1′, and 2 are the sequence numbers of the overhangs) was assembled by 2D1 and D1′ to 1280
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Figure 3. AFM characterization of LnS and LnD nanoassemblies. (A) AFM images of DTH nanoassemblies. (B) Histograms of end-to-end distance of DTH nanoassemblies in the AFM images. The red curves in B are the Gaussian fitting curves of the end-to-end distance. The mean value and standard deviation are indicated in each histogram.
the number of tetrahedron units (Figure 2A). The mobility of these structures perfectly matched the theoretical molecular size of each entity (Table S3) referring to the DNA ladder, whereas each pair of LnS and LnD with the same number of tetrahedron units showed little distinction in the mobility because the difference in bp numbers as a result of the variation in linkage style only accounts for less than 10% of total bp numbers. The structural morphology beyond those directly proportional to bp numbers, such as charges and molecular weights, are too subtle to be distinguished by the agarose gel electrophoresis. The DTH assemblies were further analyzed by size-exclusive chromatography (SEC), which is a separation method based upon the average globular size of bulk ensembles. As shown in Figure 2B, the retention times (TRs) for LnS with unit numbers of 3, 4, 6, and 8, are 12.84, 12.10, 11.37, and 11.03 min, respectively, while those for LnD are 13.21, 12.28, 11.46, and 11.13 min. Similar as that on gel electrophoresis, both families of DTH assemblies showed a shorter retention time for the 1D assembly with more DTH units, consistent with a larger molecular size in theory, whereas, all of the LnD showed a slightly longer retention time on the SEC column than that of the LnS analogues. The difference in retention time (ΔTR = TR(LnD) − TR(LnS)) declines with an increasing number of DTH, even though ΔTR for the L3 pair was already not large enough to separate L3S and L3D. However, the discrepancy in SEC retention between LnD and LnS analogue pairs, though very minimum, suggests that there could be a structural difference between LnS and LnD even after averaging over a large ensemble set. However, SEC still cannot allow us to identify the difference in the structural morphology or even
form a double linkage (11′) within the dimer and two singlestranded overhangs (2) at two vertexes of the second DTH on the desired 1D assembly. In the following chain elongation process, each step introduced a dimer (mD11′Dn, m and n are the sequence numbers of double overhangs on each DTH) to the chain as a building block unit. Finally, the 1D extension was terminated by either a single DTH or a dimer for the DTH assembly with an odd or even number of DTHs, respectively. To achieve maximum simplification in linkage design, all the dimers used the same internal double-linkage sequence, 11′. Only two pairs of linkage sequences, 22′ and 33′, are used in alternative steps for the dimer extension. Because of the fact that only one pair of linkages is exposed as a single-stranded overhang in each step, in theory, only these two pairs of duplex sequences are necessary to accomplish 1D extension with an unlimited number of DTHs, though a maximum eight DTH units in one assembly are demonstrated in this work. In total, only seven DTHs are used to assemble all LnD (Table S2). This stepwise extension strategy allows only two counterparts to participate in each isothermal-annealing step, which would ensure the hybridization efficiency. By removing one overhang from each DTH, LnS were assembled with the same step strategy. The detailed structures of each LnS and LnD are illustrated in Figure 1B and Figure S1. The preparation process was described in detail in the Experimental Section. Bulk Ensemble Characterization by Agarose Gel Electrophoresis and SEC. The successful assembly of two series of DTH nanoassemblies were verified, and the structural morphology were first characterized by agarose gel electrophoresis. The extension of the 1D nanoassembly was clearly shown by the decline in band migration along with increasing 1281
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Figure 4. Solid-state nanopore electrophoresis of L6S and L6D by a conical glass nanopore (CGN). (A) Illustration of L6D nanoassembly traversing CGN. (B) Typical current trace of L6D (top) and L6S (bottom) vs time extracted from a 1−2 h recording. (C,D) Scatter plots of current drop vs duration of DNA translocation for pure L6S (C, purple), L6D (C, orange), and a 1:1 mixture of the two nanoassemblies (D, green). (E,F) All-point current drop histograms of pure L6S (E, purple), L6D (E, orange), and the mixture (F, green). (G) A typical current trace of L6D and L6S mixture vs time extracted from a 1−2 h recording.
counted for each sample, and the distance distribution is shown in Figure 3B. If comparing the histogram of LnS and LnD with the same unit number, the average distances are similar for n = 3 or 4. For short assemblies, the linear structures still could be reached by both types of assemblies, or the backbone curling is not severe yet. However, the increment of distance from L3D to L4D (11.2 nm) is larger than that of LnS (7.6 nm), indicating that the rigid effect from the double linkage started to emerge even in the short assemblies. The difference in the average lengths is more pronounced when n reaches 6 and 8. L6D showed an ∼12 nm increment from L4D, when only a 4 nm increment was obtained for L6S. More remarkably, the distance discrepancy between L8D and L8S soared to 21.6 nm, while only a 4 nm extension was obtained from L6S to L8S. The average end-to-end distance of the LnD series escalated linearly with >11 nm elongation for every addition of a dimer unit. A near linear increment of the end-toend distance suggested that the LnD assemblies could maintain a quite high bending stiffness and behave much closer to a linear chain or rod as expected by design, while LnS series deviate ominously from the linear conformation. However, unlike 1D wireframe DNA origami, LnD assemblies still possess backbone flexibility to a certain degree. For L8D, two main peaks can be fitted to the distribution histogram. One peak was centered at 47.1 ± 6.1 nm, representing the bended structures; the other peak at 71.8 ± 16.4 nm was the population of slightly bended or linear structures. For such a long LnD assembly, the DTH chain can bend at a certain linkage position, while the rest of chain holds linear conformation as two or several short rods. Hence, AFM measurement provides a snapshot of the conformation dynamics of both DTH assemblies upon immobilizing individual structures on the surface. Both images and statistical analysis indicate that the dynamic conformation and bending stiffness of the DTH assembly are varied according to the
just isolate the two types of 1D nanoassembly, especially when the DTH number increases, and the size of 1D nanoassembly approaches the separation limitation of SEC column. Single-Molecule Characterization by AFM and SolidState Nanopore Technology. To elucidate the structural morphology between LnS and LnD, a series of measurements at the single-molecule level were carried out for both 1D nanoassemblies. First, the structural conformation of the assemblies was characterized by fluid mode AFM (Figure 3A). DTH assemblies were instantly immobilized on the mica surface by electrostatic attraction, and the conformation of individual nanoassembly molecules could be locked on a 2D surface and be visualized with nanometer resolution. The conformations of LnS molecules are apparently the softwire type. Even as short as L3S, very few linear conformations were observed in AFM image, while the majority of assembly molecules bent at the central DTH and showed a variety of curvatures. With the addition of DTH units, no linear conformation could be visualized from L4S up to L8S. LnS ensembles twisted severely with larger unit number. Random coiling conformations as a condensed cluster of DTHs emerged from L4S and became the major conformation in the AFM image of L8S. On the contrary, as shown in the lower panel of Figure 3A, many more LnD ensembles can retain their linear conformation. The majority of L3D and L4D molecules showed a short rod structure. Even in the L8D image, the linear conformation could be spotted. Many ensembles only bent at one or a couple of the DTH sites and showed an extended or “open” conformation compared to the cluster conformation of L8S. Hence, the backbone flexibility along the assembly axis is significantly reduced, and bending stiffness is enhanced in comparison to LnS structures. The bending stiffness of LnS and LnD was further analyzed statistically by measuring the end-to-end distance of individual ensembles in AFM images. At least 200 structures were 1282
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conformation dynamics resulting from the variation of linkage patterns in the DTH nanoassembly can be readily observed by nanopore electrophoresis, for either pure or mixture samples.
linkage pattern, while other properties of the assembly such as number and sequence of the DTH unit remain the same. To study the conformation dynamics of DTH nanoassemblies in solution phase, we used solid-state nanopore electrophoresis. Nanopore electrophoresis is a promising single-molecule analysis technology that can detect a wide range of species such as DNA, proteins, and small molecules.38−49 The detection principle lies in the transient current drop when an inert polymer is driven by voltage to pass through a nanopore.50 With the advantages of high-throughput capacity, size-exclusive recognition, and a completely homogeneous working condition, this technique has successfully detected a variety of nucleic acid nanostructures, such as the hybridization chain reaction (HCR) products,51 DNA cubes/ RNA rings,52 and linear/branched dsDNA.53 These recent work suggests that the nanopore technique could have the potential to distinguish a subtle conformation difference of DNA nanostructures. Here, we used a conical glass nanopore (CGN) to examine both types of nanoassemblies, since the diameter of nanopore (∼12 nm) is suitable to allow the translocation of the DTH (theoretical edge length ≈ 6.8 nm) used in our work (Figure S3). The pore size is less than twice of the spherical diameter of a DTH, which is also the nominal width of DTH 1D assemblies in the linear conformation. Four samples, L3S, L3D, L6S, and L6D, were analyzed under the same working condition. As shown in Figure 4 and Figure S4, when the DTH assembly was electrophoretically driven through the nanopore, both DTH hexamers showed a larger current drop and longer duration time than those of the DTH trimer. Both electric parameters can distinguish DTH assemblies with a substantial length difference that was due to the composition variation, such as different unit numbers of the building block. Further analysis of the electrophoretic properties between the assemblies with the same unit number revealed more subtle conformation dynamics in the solution phase. L3S and L3D showed similar current blockage, which suggests that both DTH trimers occupy the similar inner space during the translocation process (Figure S4). Meanwhile, for DTH hexamers, a significant difference in the current blockage was observed. L6S showed a current drop of 77.8 ± 7.2 pA, which was 2.6 times greater than that of L6D (29.5 ± 4.9 pA). This is consistent with the conformational features of the two structures revealed by AFM (Figure 3A). With double linkages, L6D showed a much higher possibility of maintaining a linear conformation as a nanometer molecular rod. When L6D was translocated through the nanopore, the extended conformation induced a small current drop around 30 pA, whereas the large current drop for L6S ensembles indicated L6S, because of the flexibility of the single-linkage design, was nearly dominated by the curling conformation as shown in the AFM image. Even though electrophoretic force may stretch L6S and induce a slightly open conformation, a majority of L6S molecules may still adopt a curled nonlinear conformation when they were passing through the glass nanopore. The significant difference in current blockage even allows an easy separation of the two nanoassemblies when L6S and L6D were submitted to nanopore analysis simultaneously in a mixture sample solution (Figure 4D,F,G). Two distinctive current drop peaks emerged at a similar pA as isolated L6D and L6S ensembles, respectively, which suggests no interference on the conformation dynamics occurs between two types of hexamers during the nanopore translocation process. The difference in
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DISCUSSION Two series of DTH nanoassemblies were fabricated by a stepwise isothermal-annealing protocol. There are several advantages in this assembly design. First, the stepwise assembly provides a procedure to allow stepwise extension of the DTH assembly in one dimension. With only two counterparts in each annealing step, this protocol ensures that the linear structures can be achieved with a precise number and defined sequence of DTHs, while only three sequences of duplex linkages are required. One sequence for an internal linkage of dimers and two used alternatively to link the incoming dimers is the simplest linkage sequence design we used in the present work. Second, the assembly design readily adopts variation on the tetrahedron cores. By introducing a DTH dimer or monomer with distinctive edge sequences, a sequence of DTHs can encoded into the 1D nanoassembly. Precise modification and functionalization on the edge, vertex, and linkage of the DTH can provide several layers of coding framework, which can be read as whole or separate systems. DTH assembly can provide templates for various subjects on the nanometer scale, such as inorganic nanoparticles, optical probes, molecular walkers, proteins, and antibodies, among others. Third, the assembly protocol can be extrapolated to assemble DTH dimers in two and three dimensions by introducing dual overhangs at the vertex. From one initial DTH monomer or dimer, two lines of DTH extension could progress in parallel to achieve dendritic or other complicate DTH assembled nanostructures. To manipulate the conformation dynamics of DTH nanoassemblies, we make LnS and LnD maintain the maximum similarity in the composition. Each pair of analoguess of LnS and LnD contains the same number and edge sequence of DTH units as well as the same sequence and order of duplex linkages and only differ at the number of duplex linkages between the adjacent DTHs. These two designs of 1D assembly would allow all the differences in conformation dynamics and bending stiffness to be attributed to the assembly design. Tetrahedron units are analogous to carbon atoms, and single duplex linkages are analogous to single sigma bonds. Each individual DTH in an LnS assembly can rotate freely around the linked vertex without cooperatively dragging the adjacent DTH. LnS assemblies are analogous to n-alkanes with high backbone flexibility and with little bending stiffness. On the other hand, with a second linkage between one more vertex pairs of adjacent DTHs, the connection pattern in LnD is analogous to cumulene with continuous double bonds. Instead of a softwire structure, LnD assemblies showed a worm-like shape with bending of 1D assembly only occurring to one or two linkage sites. DTHs in LnD are connected at two vertexes. The pair of double linkages is locked into one plane. The adjacent linkage planes are perpendicular to each other. The rotation of DTH restrains the adjacent double-linkage planes to 2D 360° around the DTH edge, like a door hinge. The subtle variation only in the linkage patterns results in high similarity in globular size of the DTH nanostructures. The two families of DTH assemblies showed little difference in bulk ensemble measurements, such as agarose gel electrophoresis and size-exclusive chromatography. Only with single-molecule measurements, the conformation 1283
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dynamics of individual assembly and the effects from linkage style on the conformation rigidity and bending stiffness of 1D assembly were revealed. LnS showed much lower bending stiffness compared to LnD. AFM showed that with the 1D assembly extended, fewer assemblies of LnS could stretch out in an “open” conformation, not even the mentioned linear rod shape. Whereas, unlike LnS, LnD no longer falls into the softwire style but behaves more like a worm, which curls only at a few joints and allows the entire assembly to move as several short rods connected together. The difference in conformation dynamics results in the large discrepancy of current blockage during the electrophoretic translocation of the DTH assembly through glass nanopore. The fact that large DTH assemblies with subtle differences in composition can be readily distinguished by nanopore electrophoresis even in a mixture solution could be seen as a promising method for single-molecule biosensing based upon the stimulus-responsive conformation alteration of DTH nanoassembly. A simple alteration on linkage number can manipulate the bending stiffness and consequential conformation dynamics of the highordered nanostructure without removing or scrabbling the inherent sequence of nanobuilding blocks. This approach would endow a wide variety of reactive and computing functionalities to the DNA nanostructure as a molecular device, logic gate, and structure template.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected] (F.S.) *E-mail: [email protected] (B.L.) ORCID
Fangwei Shao: 0000-0003-2007-3920 Funding
This work is supported by a Singapore AcRF MOE Tier 1 grant (M4011554), the K.C. Wong Education Foundation, China, and a start-up grant from Zhejing University. Notes
The authors declare no competing financial interest.
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CONCLUSION In conclusion, we have designed and fabricated two series of one-dimensional nanostructures with a defined number and sequence of DNA tetrahedrons. With simple variation in the number of linkages, the conformation dynamics of DTH nanostructures can be well controlled. The characterization with AFM and the solid-state nanopore technique revealed the conformational structures of LnS and LnD. Both techniques showed higher linear rigidity of LnD compared to LnS, especially for large assemblies. This is the first time that it is shown that the 1D bending stiffness of DNA nanostructures is dynamic both on the surface and in solution at single-molecule level, which may pave the way to understanding the dynamic behavior of DNA nanostructures as molecular devices and biomedical nanovessels. In addition, the work also demonstrated the power of the solid-state nanopore technique in analyzing the dynamic conformation of nanostructures and in sensing subtle conformation changes in biomolecular nanostructures. Profound studies in the future on many important factors like pore size and driving voltage may provide more definite correlation between the electrophoretic signals and nanostructures, which might open up a new era for the solidstate nanopore technique in the characterization and detection of biomacromolecules as well as tracking the reactions/ motions in nanodevices.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00834. Oligonucleotide sequences for DNA tetrahedrons, additional structural information, plots, solid-state nanopore analysis (PDF) 1284
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