Artificial DNA Nano‐Spring Powered by Protons

Abstract

A novel multifunctional, proton-fueled DNA nano-spring has been constructed. By incorporation of the G-quadruplex/i-motif sequence into the assembly, the nanodevice can perform spring-like motions in response to changes in the environmental pH without permanent deformation. Nanosized objects/functional groups could be assembled/disassembled into this system in an addressable, contractile, and reversible manner. For over 20 years, DNA has been recognized as an attractive building material in nanotechnology and materials science owing to its unique molecular recognition properties and structural features. Many DNA-based, artificial structures/devices, such as one-dimensional, two-dimensional, and three-dimensional architectures, have been constructed and shown potential applications in miniaturized biosensors, drug delivery, nanorobotics, and dynamic nanomaterials, etc.1 One-dimensional objects are unique electronically and biologically active materials. The self-assembly of small molecules into 1D nanostructures offers many potential applications, especially in the field of DNA-based nanotechnology that seeks to engineer synthetic DNA polymers to encode information necessary for the realization of desired structures or processes on the molecular level.2 Hence, the use of programmable, self-assembling DNA scaffolds appears to be one of the most promising avenues available for creating linear structures and has been proposed as a method for creating arbitrary patterns. For example, a linear DNA array displaying a switch of cis and trans conformations through the rotational motion of a robust sequence-dependent DNA nanodevice was recently demonstrated by Yan et al.3 A guanine nanowire that can be controlled by chemical input signals has also been developed.4 I-tetraplex and DNA nanotubes have also been reported for their use as building blocks for the construction of nanowires.2, 5 In addition, DNA has been designed to direct the assembly of protein and nanoparticle linear arrays by the use of appropriate attachment chemistry.1, 1, 6 Although these DNA structures/devices hold great promise in nanotechnology and nanomedicine, the creation and expansion of addressable materials with increasing patterns of complexity, molecular diversity, and further functions still remains a big challenge in this field. Herein, we describe the design and construction of a novel DNA linear structure, termed DNA nano-spring, which can perform spring-like motions with controllable functions. Our strategy is illustrated in Scheme 1. The nano-spring is prepared with two similar but not identical building blocks (subunit I, II) assembled together by a G-quadruplex forming oligonucleotide (strand D). Each subunit is formed from two single-stranded DNAs (ssDNAs): a 59mer strand C for formation of the circular molecule, which resembles the coil of the spring and a 57-mer linker (A or B) containing four guanosine-rich stretches. The circular DNA was obtained by self-ligation of the 5′-phosphated strand C through T4 ligase. Differentiated from the spiral of the spring, the nano-spring is composed of separated coils which are linked together by strand A, B, and D, respectively. The flexible linker strands (A, B, and D) were carefully designed to have one large (27 bases) region and two shorter (15 bases) identical regions complementary to coil C, so that two subunits (I and II, as depicted in Scheme 1) were produced that could readily be incorporated into a linear array in a controlled manner through hybridization with strand D. The linkers A, B, and D have four guanosine-rich stretches which ensure the coils to be separated evenly and the coil can, thus, be lengthened by hybridization with the complementary cytosine-rich strand E. As can be seen in Scheme 1, operation of the nano-spring is powered by a proton. Under slightly acidic conditions, the cytosine residues are partially protonated and the DNA folds into a closed i-motif structure.7 Meanwhile, the linker domains remain quadruplex structures and lead the nano-spring to adopt a compressed state. When the pH is increased to the alkaline region, the cytosine residues are deprotonated and hybridized with the G-rich complementary strand to form a rigid duplex. The cooperative hybridization of all subunits translates into an overall extension of the nano-spring, allowing it to adopt an extended state. Therefore, it can reversibly perform spring-like motions in response to environmental cues. The length difference between the extended and compressed state is the working length of the spring. More importantly, unlike a real spring, the proton-fueled DNA nano-spring does not suffer from permanent deformation. Furthermore, the assembly could be utilized as a functional smart material to immobilize nano-sized objects with a precise relative spatial orientation in a robust, programmable, and controllable fashion. To the best of our knowledge, this is the first report of assembling/disassembling of nano-objects in an extended DNA array with contractile, reversible features. Schematic representation of the construction and operation of the proton-fueled DNA nano-spring. The flexible linker strands A, B, and D were designed to have one large region and two shorter identical regions complementary to coil C, so that two subunits (I and II) were produced that could readily be incorporated into a linear array in a controlled manner through hybridization with strand D. A G-quadruplex/i-motif sequence containing four stretches of GGG/CCC was incorporated into strand A, B, D, or E to enable the nanodevice to perform spring-like motions in response to pH stimulus. Gel-electrophoresis experiments were carried out first to confirm the assembly and the operation of the DNA nano-spring. The 5′-phosphated C strand was circularized using T4 ligase and purified by 12% denaturing gel (Figure S1). A stoichiometric amount of each DNA strand was then mixed at a concentration of 5 μM in tris-acetate-ethylenediaminetetraacetic acid (TAE)/Mg buffer. The mixture was heated to 95 °C for 10 min and then cooled to 20 °C over 48 h and incubated at 4 °C for at least 10 h. As shown in Figure 1A, subunits I and II migrated as a clear sharp band with expected gel mobility (lane 2 and 3). The assembled DNA structure, which was designed to adopt a compressed state (a combination of subunit I, II, and strand D), migrated much more slowly (lane 4) and stayed near the baseline. The broad band indicates both its high molecular weight and the polydispersity reflectivity of the polymeric material. Further addition of strand E (i-motif DNA) into the mixture resulted in the slowest gel-shift mobility (lane 5), signaling the creation of a species with higher molecular weight corresponding to the extended state of the nano-spring. Since the gel-shift mobility of these two states could not be distinguished well by polyamide gel electrophoresis (PAGE) at pH 8.0 and 6.0 (Figure 1A), additional agarose gel electrophoresis was carried out. As illustrated in Figure 1B, the assembly of multiple repeats of subunit I and II resulted in smeared bands because of the inhomogeneous molecular weight (lane 4 and 5). At pH 8.0, smeared bands corresponding to a length of about 1000 bp (Figure 1B, lane 4) and 3000 bp (Figure 1B, lane 5) developed in the absence and presence of strand E, respectively, which can be clearly seen when comparing with the linear molecular weight standards (lane 6). The slower mobility shift indicated the formation of higher molecular assemblies through the formation of the rigid duplex in the linker domain. Interestingly, when the pH was adjusted to the slightly acidic region (pH 6.0), the assembly migrated to the same extent in the absence or presence of strand E (Figure 1B, lane 4 and 5), indicating that the linker domain stayed in a G-quadruplex structure upon the formation of the closed i-motif DNA. Native gel electrophoretic analysis (A: PAGE, B: agarose) of the DNA nano-spring at pH 8.0 and pH 6.0. The contents of the lanes are listed as follows: Lane 1, circular strand C; lane 2, subunit I; lane 3, subunit II; lane 4, assemblies without strand E (compressed state); lane 5, assemblies with strand E (extended state); lane 6, molecular weight marker. (100–3000bp. 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 3000.) To gain further support for the proposed design and construction, direct observation of the inter-conversion of the nano-spring was demonstrated by atomic force microscopy (AFM). In our system, the different states of the nano-spring are attributed to the change in distance between adjacent coils. Specifically, the extension and contraction is modulated by the conformational changes between quadruplex and duplex DNA. At pH 8.0, the designed 27-mer i-motif sequence can hybridize with the corresponding sequence on the A, B, and D strands, and form a fully stretched duplex about 9.2 nm (27 × 0.34 nm) in length. Therefore, the length of each basic building block formed by subunit I and II is around 27.6 nm (3 × 9.2 nm) in the presence of strand E. A typical AFM image of the assembly shows linear structures with periodic dots decorated on it (Figure 2a). (The flexible coils appear as dots because of the deformation and the well-known limitation of the lateral resolution of the microscope.8) The average distance measured between two adjacent coils was about 10 nm, matching the designed distance of a fully stretched 27-mer duplex (AE, BE, or DE). Moreover, the measured distance of each basic building block (I-DE-II) was about 31 nm, which is also in agreement with the calculated value from the model. Both measured values were slightly larger than the designed distance of a fully stretched 27-mer duplex (AE, BE, or DE). This slight deviation can be attributed to surface tension.9 The height of the assembly in the extended state was 1.2 nm and the width of the assembly was about 19 nm, which is consistent with that of duplex DNA. The observed length of the 1D wire in the extended state was in the range of 1 μm to 3 μm (Figure S2). When the pH was lowered to slightly acidic conditions (pH 6.0), strand E dissociated from the duplex, and the G-rich sequence in the linker region formed a compacted G-quadruplex with a length of around 1.5 nm. Similar to the coils, the quadruplexes are also visualized as dots. In this case, coils and quadruplexes could not be differentiated distinctly and linear structures were observed for the assembly (Figure 2b). The height of the 1D wire in the contracted state was 0.5 nm and its width was about 12 nm (Figure S2). The interspacing was obtained by taking the cross-sectional data and plotted these in a histogram. A smaller interspacing of 6 nm compared to that under basic conditions was measured. The average length of the assembly in the contracted state was about 0.9 μm. Interestingly, the reversible switching of the nano-spring could also be observed by the AFM measurements. As shown in Figure 2c, linear structures with wider spaced dots were visualized again when the pH value was increased to 8.0. To further support the interspacing change between each unit, the linker strands A, B, and D were lengthened to eight guanosine-rich stretches. At pH 8.0, the interspacing was measured to be 21 nm (Figure 2d), which is comparable to the calculated value. The small deviation could be a result of surface tension. Thus, the AFM results confirmed the gel-electrophoresis findings and unambiguously showed that the DNA nano-spring could perform extension and contraction as expected: a compressed state and an extended state are shown in smart response to pH stimulus. a–d) AFM images in height-mode (top panels) of the DNA nano-spring. The scale bars are 100 nm. Cross-sectional profiles (left) and histogram (right) of the distances between the subunits are shown in the bottom panels. The black arrows at corresponding sites in the bottom left panels represent the peaks of the periodical dots. In the bottom right panels, “f” and “d” stand for the frequency and distance (nm), respectively. Two hundred measurements were taken for each state in the statistical analysis. The insets show magnified images in phase-mode, the scale for each of these images is shown at the bottom. One individual unit on each image is marked with a white bracket. One duplex stretch is marked with white arrows in Figure 2d. a) Extended state. b) Contracted state. c) Re-extended state. d) Extended state of the assembly with eight guanosine-rich stretches. The real time motion of the nano-spring was then monitored by Förster resonance energy transfer (FRET) experiments. We labeled the linker D strand with a fluorophore (fluorescein) and a quencher (tetramethylrhodamine) at its 5′ and 3′ ends, respectively. The distance between the two dyes was expected to increase in the extended state and decrease in the compressed state. A working pH of 6.0 was chosen to minimize the pH influence to the fluorescence signal. As can be seen in Figure 3A, in the compressed state at pH 6.0, the fluorescence signal of fluorescein was of low intensity. While in the extended state at pH 8.0, the fluorophore was well separated from the quencher, the energy-transfer efficiency was low, and the fluorescence intensity was strong. As shown in Figure 3B, we chose two intermediate states to demonstrate the FRET process. Upon a small pH increment, the fluorescence intensity around 520 nm was observed to increase and that around 575 nm was observed to decrease. The FRET process was further confirmed by a spectral change after the addition of strand E at pH 8.0 (Figure S3). Importantly, the nano-spring can continuously cycle between the two states when the solution pH oscillates between 6.0 an 8.0. The cyclical changes in fluorescent signal at 520 nm that resulted from the extension and contraction of the system are shown in Figure 3C. Since the waste products are water and NaCl, which would not interfere with the system, the nano-spring shows no apparent loss in efficiency after 10 full operation cycles. The response of the system is crucial for its operation and potential applications. In this system, the response is very fast with a switching time of 39 s. Although it is slower than that of a single molecular proton-propelled machine (5 s),10 it is at least 1–2 orders of magnitude faster than that of a DNA-fuelled11 or chemical oscillator-filled system.12 Furthermore, a control experiment was carried out using a non-bridged D/E homogeneous system. A faster switching time of 15 s accompanied by a smaller fluorescence intensity change was observed (Figure S3). The different kinetic and response values provide further evidence that a DNA nano-spring is indeed formed that can be oscillated between two well-defined states in a pH-dependent manner. A) Comparative fluorescence spectra of the DNA nano-spring at pH 8.0 and 6.0 (excitation at 497 nm). B) Comparative spectra of the assembly at pH 6.5 (black) and 7.0 (gray). The spectra were obtained through excitation at a wavelength of 497 nm (fluorescein) and 549 nm (TAMRA), respectively. C) Cyclic activation of the nano-spring between the extended and contracted state at different pH cycles. The kinetics of the fluorescence intensity changing with time were measured at an excitation wavelength of 497 nm, and monitored at a wavelength of 520 nm. DNA-mediated assembly of nano-sized objects provides an attractive way to organize protein, fluorophore, metallic, and semiconducting nanoparticles into periodic or discrete 1D, 2D, and 3D architectures.1, 2, 6, 8, 13 However, the fabrication of well-extended, functional nanoparticle arrays in a precise and controllable manner remains largely unexplored. An important element in our design is the long contractile DNA scaffold, and we report here the first example of precisely assembling/disassembling nano-objects in an extended DNA array with contractile, reversible features in response to environmental cues. To demonstrate the contractile motion of the nano-objects, for example, two different fluorophores were tethered at the 5′ and 3′ ends of strand D, respectively. As illustrated in Figure 4A, the as-prepared array allowed the attached multi-copy fluorophores to be continuously cycled in a close or separated state and FRET was accomplished in a pH-dependent manner (Figure 3C). On the other hand, the controllably assembly/disassembly of nano-sized objects along the DNA nano-array was demonstrated by attaching 5 nm citrate-coated gold nanoparticles (AuNPs) to the thiol-modified 5′ end of strand E. As illustrated in Figure 4B, the self-assembly of into 1D arrays was obtained through DNA hybridization in the reversible assembly/disassembly of nanoparticles along the DNA was in with the extension and contraction of the The assembly and operation was also by Figure shows that the 1D DNA array was with gold nanoparticles at pH 8.0. In when the pH value of the system was to 6.0, evenly spaced could not be indicating that the i-motif DNA dissociated from the microscopy further the of the by the DNA The images in Figure show that at pH 8.0 the were evenly spaced on the DNA array because of hybridization of strand E with linker strand D. However, no was observed for the at pH 6.0 (Figure Importantly, the periodic when the pH value of the system was to 8.0 (Figure As can be seen in the the distances between two adjacent is about 10 nm, which is in agreement with the AFM The AFM and results as complementary evidence along with the gel-electrophoresis and FRET to clearly demonstrate the use of this system for functional and materials. Schematic of the incorporation of nano-sized objects into the system and assembly/disassembly of nanoparticles on the DNA array in a contractile and reversible manner by C) of of on the DNA The insets in and are the of the distances between the In we have shown a novel strategy to a DNA nano-spring powered by The nano-spring was prepared by circular DNA and G-quadruplex/i-motif forming and it has two important properties that it for the of DNA-based functional materials. the incorporation of the G-quadruplex/i-motif sequence into the assembly allowed the to perform spring-like motions in response to the environmental This of DNA nano-spring is robust and reversible without the of a real spring, the proton-fueled operation does not suffer permanent deformation. The response of our is at least 1–2 orders magnitude faster than that of a DNA or a system and it does not duplex waste products that the system. and more importantly, identical or different objects/functional such as and could be incorporated into this system with precise Our demonstrated the first example for the assembly/disassembly of nano-objects in an extended DNA array with addressable, contractile, and reversible features. materials are important for many applications, and this remains largely unexplored. This one in this The of this system it attractive for drug delivery, such as conditions that are by their high Furthermore, the as well as the be by changing the linker DNA such by increasing the guanosine-rich Although G-quadruplex/i-motif was as a system to show the construction and operation of the nano-spring, many other G-rich DNA that in and in many processes could be and DNA with motions and could be constructed through Therefore, this is an important in DNA-based with and and be for and applications. and was from was from T4 DNA ligase and T4 were from DNA was from the other in the experiments were from the DNA were from and purified by The concentration of each DNA sequence was by at nm. The were by the DNA here are listed in The experiments were carried out on gel and for h with a field of at 4 The of pH 8.0, acid and were with electrophoretic experiments were on agarose gel in 0.5 × at for 2 h at 4 The were with AFM was in the on a using using a in 10 of the DNA assembly was to a of was left to to the surface for 5 water was to the on the to the was with compressed were on a at 100 The for were prepared by of the a which was then in a measurements were carried out on using a of 1 at an excitation wavelength of 497 nm (fluorescein) or 549 nm spectra were monitored from to nm and from to nm, respectively. The for the excitation and were both to 5 nm. The kinetics of the fluorescence intensity changing with time were measured at an excitation wavelength of 497 nm, and monitored at a wavelength of 520 nm. the addition of or the solution was mixed 4 s by it with a and measurements were at 20 of The 5′ ends of strand C were by of strand C with T4 at °C for 3 h in which pH and the was heated to °C for 10 min to the and cooled slowly the a of T4 DNA ligase was The were incubated at this for about h and the was at 95 °C for 5 The formation of circularized DNA was by (Figure S1). the circularized strand C was purified by 12% denaturing of The stoichiometric of each strand in the unit was at a concentration of 5 in pH 8.0, acid and The solution was cooled slowly from 95 °C to 20 °C over 48 h and incubated at 4 °C for at least 10 h. of gold nanoparticles were and an amount in of DNA was to the solution of The solution was at the solution was into a (pH and allowed to stand for by for min at 13 to of the the was with a (pH and in (pH The solution was at 4 °C is available from or from the The are for the support of the of and the from the of of to are as are but not or are available as by the The is not for the or of information by the than be to the corresponding for the