Manipulation of DNA Origami Nanotubes in Liquid Using Programmable Tapping- Mode Atomic Force Microscopy

Manipulation of DNA origami nanotubes in liquid using programmable tapping- mode atomic force microscopy

Longhai Li1,2, Xiaojun Tian1, Zaili Dong1, Lianqing Liu1, Osamu Tabata3,4, Wen J. Li1,5 1State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang, People’s Republic of China 2University of Chinese Academy of Sciences, Beijing, People’s Republic of China 3Department of Micro Engineering, Kyoto University, Kyoto, Japan 4Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany 5Deparment of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong, People’s Republic of China E-mail: [email protected]

Published in Micro & Nano Letters; Received on 15th August 2013; Accepted on 21st August 2013

The nanoscale manipulation of 1D soft and ﬂexible ‘DNA origami nanotubes’ (DONs) that are 6 nm in diameter and 400 nm in length, and placed on a mica substrate in a TE/Mg2+ buffer solution, was executed by an atomic force microscopy (AFM) tip using a programmable tapping-mode manipulation strategy. The AFM’s tip tapping amplitude was controlled to be less than or equal to 10 nm while vibrating in a solution with Mg2+ concentration of 10–20 mM. A series of single-point and one-step manipulation experiments revealed that manipulation can be achieved with an 80% successful rate under the condition that the AFM tip vibration amplitude is 3–4 nm and the Mg2+ concentration is 10 mM. Utilising this optimised condition, multipoint and multistep manipulation based on the programmable tapping-mode AFM process was conducted and nanomanipulation of DONs was successfully demonstrated.

1. Introduction: Deoxyribonucleic acid (DNA) origami [1] has during manipulation using a force model between the tip and attracted attention as an excellent platform to arrange various sample’s surface. Prior to Xi’s work, Ritter et al. [14] also nanomaterials, such as metallic nanoparticles, carbon nanotubes demonstrated the pushing of antimony particles on a graphite (CNTs) and so on [2–4], precisely using the base sequence surface using contact-mode manipulation. However, manipulation information of a scaffold DNA. Moreover, DNA origami approaches based on contact-mode have the drawback of wearing can bind with each other with the help of hybridisation of out the AFM tip easily as the contact force is significant, which DNA strands arranged at its edges and form higher-order makes continuous nanomanipulation difficult. Furthermore, it is structures of micrometre-scale dimensions [5]. Based on these difficult to obtain clear nanoscale images after contact-mode features of DNA origami, we proposed to use DNA origami as manipulation, especially for DNA molecules in liquid which nanoscale functional building blocks and place them on needs sharp AFM tips in order to obtain good image quality. microelectromechanical systems (MEMS) to realise nano/ Hence, tapping-mode manipulation was proposed to solve this microengineered and molecular systems (NEMS) [6]. The problem and was found to have a higher success rate of nanoscale concept of this proposed technique is termed as programmable manipulation and imaging. Resch et al. [15] utilised the tapping- oriented self-assembly on MEMS (P-OSAM) [7] and is illustrated mode AMF tip to push a nanoparticle by decreasing the vibration in Fig. 1. As shown, a micrometre-scale functional structure is set point, while the Z-feedback was turned off during the manipu- integrated on a MEMS substrate by the oriented self-assembly of lation process. Liu et al. [16] developed a new tapping-mode-based multiple nanoscale functional building blocks made of DNA AFM manipulation method, which can push nanoparticles by chan- origami functionalised by nanomaterials. To accomplish this ging the amplitude set point, scanned area, aspect ratio of the concept, the placement of the initial DNA origami as an origin of scanned area and the scanning angle. However, their method has the subsequent oriented self-assembly at the desired position is a been only applied for particles made of hard (i.e. low elasticity) critical process. In this Letter, we propose a new approach materials. To the best of our knowledge, no one has attempted to utilising the programmable tapping-mode atomic force microscopy (AFM) manipulation technique developed by our group [8, 9] to place the initial DNA origami in a desired position in a fluidic medium. Atomic force microscopy (AFM) [10] has become the most popular tool for researchers in the research areas of nanoimaging and nanomanipulation. It can be applied not only for imaging and studying the surface topography in nanoscale, but also for the manipulation, assembling and cutting of nanoscale objects. After the first demonstration by Schaefer et al. [11] and Junno et al. [12] in 1995, AFM-based nanomanipulation has been widely used by Figure 1 Illustration of the proposed programmable oriented self-assembly many groups. Many kinds of nanoscale objects were manipulated on MEMS (P-OSAM) concept or assembled into special patterns in recent years. For example, a Initial DNA origami is manipulated to a desired position by using a ’ programmable tapping-mode AFM tip on MEMS substrates Xi s group [13] pushed latex particles and silver nanorods on a b Assembly of AuNPs decorated DNA origami is performed by oriented glass surface using their augmented reality-based AFM by contact- self-assembly mode, which could display real-time changes in the environment c Plasmon waveguides below the diffraction limit of light can be constructed

Micro & Nano Letters, pp. 1–5 1 doi: 10.1049/mnl.2013.0483 & The Institution of Engineering and Technology 2013 manipulate soft and flexible material with a length of a few hundred nanometres such as DNA origami nanotubes (DONs) in liquid. To address this task, multipoint and multistep manipulation is required, which is implemented by the programmable tapping-mode AFM manipulation method [8, 9] with optimised manipulation parameters for DONs. As a first step to verify the feasibility of using the programmable tapping-mode AFM manipulation method (i.e. where the AFM tip’s trajectory, vibration amplitude and moving speed are controlled by Figure 3 Conceptual illustration of the programmable tapping-mode AFM manipulating method an algorithm) to manipulate DONs in a fluidic medium, compre- hensive experiments were conducted and the results are presented in this Letter. The effects of two important manipulating parameters – method plans the trajectory, the manipulating amplitude and the manipulation amplitude of the AFM tip and Mg2+ concentration velocity of a tip by a program. The AFM tip vibrates during of the fluidic medium – on the nanomanipulation results are imaging and manipulation with a pre-determined amplitude presented, including the optimised values of these parameters as similar to the typical operation of the AFM tapping-mode determined experimentally. Section 2 describes the sample scanning method. The manipulation work reported here is preparation and the AFM nanomanipulating method. Section 3 conducted according to the following steps. (i) Deposit 2 μl summarises and discusses the experimental results. DONs-dispersed buffer solution on a mica surface, wait for 5 min and then scan the sample with the AFM tip in the buffer solution. 2. Material and experimental method (ii) Choose a target DON to be manipulated from the scanned 2.1. DON and AFM: The size of the DONs used in the experiments image. (iii) Set values of tip displacement, distance of shown in Fig. 2e is 6 nm in diameter and 400 nm in length as manipulation and vibration amplitude and speed. (iv) Execute reported by Kiss et al. [17]. The TE/Mg2+ (20 mM Tris-HCl, pH the manipulation process. (v) Scan the sample again to check the manipulation result. Since the AFM tip knocks the sample 7.6, 1 mM EDTA, 10–20 mM MgCl2) buffer solution containing DON were deposited onto freshly cleaved mica. Imaging and surface intermittently during the tapping-mode operation, the time manipulation were both conducted by a tapping-mode AFM of tip-to-DON contact is shorter than if contact-mode operation system (Digital Instrument Nanoscope III) which is a Bruker were used. Therefore damage to the tip and the sample can be Multimode AFM with a E scanner head and DNP tip. reduced, which is crucial for manipulating soft materials in liquid while keeping a sharp AFM tip for high resolution imaging. 2.2. Manipulation procedure: Fig. 3 illustrates the trajectory of an AFM tip during the programmable tapping-mode AFM 2.3. Manipulation parameters: As mentioned previously, in this manipulation. The programmable tapping-mode manipulation Letter, the concentration of Mg2+ of a buffer solution and the

Figure 2 Structure of the DNA origami nanotubes used in our experiments a DONs used are 6 nm in diameters b 28 repeating staple pattern units c M13mp18 scaffold path d DNA staple pattern e AFM image

2 Micro & Nano Letters, pp. 1–5 & The Institution of Engineering and Technology 2013 doi: 10.1049/mnl.2013.0483 manipulating amplitude were found as two important parameters to be optimised, which have a strong influence on the successful rate of manipulating DONs in a fluidic environment. To manipulate a DON successfully on a substrate in the buffer solution, the attraction force should be optimised to prevent the samples from floating into the solution because of the weak binding force or stiction of the sample to the substrate caused by strong binding force. DONs are ‘adsorbed’ on the mica surface in a liquid environment because of the existing Mg2+ between DNAs and the mica substrate that acts as the ‘salt bridge’. When the concentration of Mg2+ in a buffer solution is increased, the fractional surface density of the divalent Mg2+ cations increases, which results in the increase of attraction force between DNAs and the mica surface, as demonstrated by the simulations [18] and experiments [19]. Therefore the attraction force between DONs and mica can be changed by varying the Mg2+ concentration in a buffer solution. Hence, the effect of Mg2+ concentration was investigated experimentally as a parameter to be optimised. In the experiments, we found that DONs would be deformed by the AFM tip during the nanoimaging process when the concentration of Mg2+ is less than 9 mM, and manipulation results are not affected by the concentration of Mg2+ when it is more than 20 mM. Hence, we focused on using the concentration of Mg2+ ranging from 10 to 20 mM with 2 mM increments in our investigation. Another important parameter affecting the DONs manipulation results is the manipulation amplitude of the AFM tip. In the manipulation experiments reported in this Letter, the tip’s horizontal speed and the tip’s moving displacement were set to 500 nm/s and 150 nm, respectively. The tip’s vibration frequency was 8.5 kHz and initial vibration amplitude was 50 nm p-p. Hence, the maximum vertical tip speed is 1 mm/s, which is 2000 times faster than the Figure 4 AFM images of the typical manipulation results: ‘bending’ in b, horizontal speed. Owing to this fast vertical tip speed, when a ‘cutting’ in d, ‘disappearing’ in f and ‘no change’ in h nanoobject under manipulation is stiff enough that it does not get deformed by the tapping force of the tip, nanomanipulation is possible even when the tip’s amplitude is larger than the height of the nanoobject. However, since DNA is a soft material with Young’s modulus of 340 MPa [20], it is easily deformed when a tapping tip contacts it. Hence, the overall diameter of some DONs may be reduced during the manipulation process because of the deformation of the DNA. Consequently, at times, the AFM tip may bypass the top of a DON even though the tip vibration amplitude is set lower than the DON’s diameter. Also, a DON may be cut if the force applied on it when the AFM tip passes through it is large enough. Therefore the manipulation amplitude is also considered as a very important parameter to be optimised in order to achieve satisfactory results for manipulating DONs. A parametric study was performed where the manipulation amplitude was varied from 0to10nmwith1nmstepsbyloweringtheAFMtip’spositionto the mica substrate. The manipulation experiments were conducted 10–12 times for each experimental condition with different amplitude and Mg2+ concentration, and the manipulated results were observed by the same AFM system and tip used for manipulation.

3. Results and discussion 3.1. Single-point manipulation experiment 3.1.1 AFM imaging results before and after the manipulation of DONs: The typical AFM images of the DONs before and after manipulation are shown in Figs. 4a, c, e and g (before) and Figs. 4b, d, f and h (after), respectively. The DONs shown in Figs. 4a, c, e and g were manipulated at the point indicated by the white arrow. The manipulation results can be categorised into four cases, that is, (i) deformation (Fig. 4b), (ii) dissection (Fig. 4d), (iii) removal (Fig. 4f ) Figure 5 Percentage of deformation, dissection, removal and no change 2+ and (iv) no change (Fig. 4h). The observed manipulation results are From experimental results of different Mg concentrations varying from 10 summarised in Figs. 5a and b. The height (diameter) of the DONs to 20 mM, the most successful deformation result was obtained at 10 mM (as shown in Fig. 5a) may be reduced during the manipulation process. However, since When the AFM tip’s manipulating amplitude was varied from 1 to 10 nm, the DONs are elastic, their original height could be restored after the most successful deformation result was obtained when 3–4nm the manipulation process. Hence, the manipulation process has no vibration amplitude was used (as shown in Fig. 5b)

Micro & Nano Letters, pp. 1–5 3 doi: 10.1049/mnl.2013.0483 & The Institution of Engineering and Technology 2013 overall inﬂuence on the height of the DONs, that is, the height of a DON before manipulation is 5.1 nm as shown in Fig. 4a, and it is also 5.1 nm after manipulation as shown in Fig. 4b.

3.1.2 Effect of Mg2+ concentration: Fig. 5a shows the effect of Mg2+ concentration using a manipulation amplitude of 4 nm, which is slightly lower than the DON diameter. The experimental results were categorised into four cases as described before. The number of the observed deformation cases decreased with increasing Mg2 + concentration. A noteworthy observation is that, when the Mg2+ concentration is above 15 mM, deformation of the DONs was not observed. On the other hand, the number of dissection cases increased with increasing Mg2+ concentration. This can be explained as DONs tend to stay at the original position when the adhesion force to the substrate is increased. In this case, the DONs could not be manipulated when the AFM tip just hits the upper part of the tubes. We note here that the force applied from an AFM tip to the DONs was estimated to be in the order of 20 nN from analytical calculations [21]. Also, Bustamante et al. [20] reported that the tensile strength of DNA is in the order of 1 nN. Hence, if we assume that the shear strength of DNA of DONs is in the same order of magnitude of the tensile strength of DNA, then the applied force to the DONs by the AFM tip should be strong enough to overcome the shear strength of the DONs and dissect them. Moreover, during experiments, deformations of the DONs were often observed just by scanning for AFM imaging at Figure 6 Manipulating DONs successfully using programmable tapping- 2+ mode AFM manipulating method aMg concentration lower than 10 mM. This means that the posi- ‘ ’ 2+ Results of the single-point and one-step manipulation process are shown in tion and orientation of the DONs are hard to control if the Mg Figs. 6a and b concentration is below 10 mM because of the weak adhesion Results for ‘multipoint and multistep’ manipulation process are shown in force between the DONs and the substrate. From these results, we Figs. 6c–f concluded that the 10 mM Mg2+ concentration is an appropriate manipulation parameter. are shown in Figs. 6d and f by the white rectangles. The DON was 3.1.3 Manipulation amplitude: Fig. 5a shows the effect of manipu- pushed about 400 nm from left to right as shown in Fig. 6d, and lation amplitude at 10 mM Mg2+ concentration. The experimental about 80 nm as shown in Fig. 6f. From these results, the feasibility results were also categorised into four cases. From Fig. 5b,itwas of using the programmable tapping-mode AFM manipulation concluded that the manipulation amplitude required to consistently method for manipulating soft and flexible 1D DONs in a liquid realise deformation of the DONs was about 3–4 nm. The ratio of environment was demonstrated. DONs removal (i.e. DONs floating in fluidic medium after manipulation by the AFM tip) against DONs adhering to the substrate 4. Conclusion: In this Letter, we report the successful application increased when the AFM’s tip vibration amplitude decreased to of the programmable tapping-mode AFM method to manipulate below 2 nm, and became maximum when the AFM tip contacts DONs in a liquid environment. The experimental results proved the mica substrate consistently (i.e. zero-amplitude tip vibration). that DONs can be manipulated using an AFM tip amplitude of 2+ Also, when the AFM tip’s vibration amplitude was above 8 nm, 3–4 nm and Mg concentration of 10 mM in a buffer solution. that is, slightly larger than the diameter of the DONs, most DONs To the best of our knowledge, this is the first time that a soft did not change their position. This means that the tip passed nanoscale material in a liquid environment has been successfully above the DONs and failed to manipulate them. Hence, a tip manipulated on a substrate. As a next step, the efficiency of the vibration amplitude of 3–4 nm was considered as an optimised proposed manipulation method based on a programmable manipulation amplitude to manipulate the DONs discussed in this tapping-mode AFM will be improved by quantitative analyses of Letter. We note here that a possible reason why the percentage of the manipulation mechanism, and the proposed methodology will dissection for 20 mM Mg2+ concentration is lower than for 15 mM be applied to other soft nanomaterials. (as shown in Fig. 5a) could be attributed to an initial deformation of the DONs at high Mg2+ concentration because of the stronger 5. Acknowledgments: This project is partially funded by the adhesion force as discussed in [20]. However, this speculation National Natural Science Foundation of China (NSFC project no: needs to be further investigated in future. 51005230) and by the China Postdoctoral Science Foundation (project no: 2012M520654). The authors thank the Croucher 3.2. Programmable tapping-mode AFM manipulation results: Foundation (Hong Kong) for providing partial funding to this According to the parameters optimised above, the manipulation project (through the CAS-Croucher Funding Scheme for Joint experiments of DONs were conducted at the condition of 3–4nm Laboratories; project no. 9500011). (For DNA self-assembly, AFM tip vibration amplitude and in 10 mM Mg2+ concentration contact: [email protected]. For Programmable AFM buffer solution. Typical experimental results are shown in Fig. 6. nanomanipulation: contact: [email protected]). The percentage of the deformation case was almost 80% in the experiments of ‘single point and one step’ manipulation process 6 References as shown in Figs. 6a and b. 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