This research was partially supported by the Global Research Development Center Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science and ICT (MSIT) (2015K1A4A3047345) and Nano&#183;Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT) (2012M3A7A9671610). The Institute of Engineering Research at Seoul National University provided research facilities for this work. Authors acknowledge gratitude towards Tae-Young Yoon (Biological Sciences, Seoul National University) regarding the fluorescence spectroscopy for the FRET analysis.

1. Design of the 6 by 6 DNA Accordion Rack with caDNAno14
<ol>
	<li>Download and install caDNAno 2.0 software14 to design a DNA accordion rack (caDNAno 2.5 is also available on https://github.com/cadnano/cadnano2.5). Open caDNAno14 and click the Square Tool to add a new part with a square lattice.</li>
	<li>Number each beam of the accordion rack and draw on the left lattice panel of the caDNAno14 (Figure 2).</li>
	<li>Click the pencil tool and draw each beam on the right edit panel on the caDNAno14. Break beams every 32 bp, which is for joints between adjacent beams. Place staple crossovers in the same position as the joints. Use the Insert Tool and the Pencil Tool in caDNAno14 to let the joints have additional single-stranded crossovers.</li>
	<li>Click the Pencil Tool and connect the joints. Each beam has seven joints.</li>
	<li>Generate scaffold crossovers to merge the scaffolds into the single loop by using the previously reported scaffold routing algorithm11. Do not let the minimum binding domain between scaffold and staple strands be less than 8 bp (Figure 3).</li>
	<li>Place the scaffolds that are not used in the assembly at the vertices located on opposite sides of the accordion rack, as shown in Figure 3.</li>
	<li>Click the Break Tool. Break the strands where staple strands are circular or longer than 60 bp.</li>
	<li>Design the DNA lock strands.
	<ol>
		<li>Click the Break Tool. Break 8 bp of a staple DNA region to make a sticky part and delete 8 bp of a staple DNA region. There are 18 sticky parts (Figure 1) in the 6 by 6 accordion rack.</li>
		<li>Place sequences that are reverse complementary to the sticky parts at both ends of lock strands and connect them by a bridging region, which consists of poly T strands of the desired length (Figure 1B).</li>
		<li>For the reconfiguration, add 8 bp of toehold sequences at the end of DNA locks for strand displacement. The toehold sequence used is in Table 2.</li>
		<li>Prepare poly A strands which are reverse complementary to the bridging region.</li>
		<li>Design strands that are reverse complementary to the DNA locks for the reconfiguration experiment.</li>
	</ol>
	</li>
	<li>Click the Sequence Tool and click scaffold DNA. Choose the scaffold as standard M13mp18. Click the Export Tool and save sequence in csv format (Table 1).</li>
</ol>
2. Simulate the Structure with the oxDNA
<ol>
	<li>Download and install the oxDNA16,17. The latest source code is available on https://sourceforge.net/projects/oxdna/files/.</li>
	<li>Make starting configuration files from the caDNAno14 file using python script &#39;cadnano_interface.py&#39;, which is provided in the oxDNA16,17 package. The usage is as follows: &#8216;python cadnano_interface.py cadnano_file.json sq&#8217;. The topology file and configuration file are now generated. 
	Note: The topology file includes how many strands and nucleotides are in the structure and information regarding backbone-backbone bonds between nucleotides. The configuration file includes general information such as timestep, energy, and box size. Orientation information such as position vector, backbone-base vector, normal vector, velocity, and angular velocity of nucleotides is also included (Figure 4).</li>
	<li>Change the information in the topology and configuration file from caDNAno14 to make them reflect the real structural information of the accordion rack. All beams are arranged in parallel when the topology and configuration files from caDNAno14 are visualized. However, the accordion rack is a lattice structure so the distance between bonded nucleotides are far for simulation (Figure 5).
	<ol>
		<li>Rotate and move each beam to the desired lattice structure. The nine columns on the left in the configuration file are the position vector, backbone-base vector, and normal vector (Figure 4). To rotate a beam, rotate all of position, backbone-base, and normal vectors using rotational transformation. Then move a beam by changing the position vector to locate it as shown in Figure 5.</li>
		<li>Relax the structure using the script provided in the oxDNA package (see example in $oxDNA/EXAMPLES/RELAX_INITIAL_CONFIGURATION for further information).</li>
	</ol>
	</li>
	<li>Run molecular dynamics simulation for ten million steps using the relaxed configuration file. The usage is as follows: &#8216;./oxDNA &#60;input&#62;&#8217; Save data every 5000 or 10000 steps.</li>
	<li>Visualization 
	Note: The structures were visualized using cogli.
	<ol>
		<li>Download and install the latest version of the cogli (https://sourceforge.net/projects/cogli1/).</li>
		<li>Run the cogli with the topology and configuration files from the oxDNA simulation. The usage is as follows: &#8216;./cogli1 -t &#60;topology file&#62; &#60;configuration file&#62;&#8217; .</li>
		<li>Hide the box by pressing b.</li>
	</ol>
	</li>
</ol>
3. Synthesis of the Structure
Note: The synthesis method is adapted from the previous protocol15,18.
<ol>
	<li>Purchase the designed DNA staples from an oligonucleotide provider.</li>
	<li>Adjust the concentration of these DNA staples to 100 &#956;M using nuclease-free water.
	<ol>
		<li>Pool each DNA strand that constitutes a &#8216;free state&#8217; structure into one tube and adjust the concentration to 2 &#956;M for each strand.</li>
		<li>Pool DNA lock strands by length and number of lock sites into tubes and adjust the concentration to 2 &#956;M for each strand. 18, 9, and 4 lock sites are used. Add poly A strands which are complementary to the bridging region at the same concentration.</li>
		<li>Pool strands that are reverse complementary to the DNA lock strands by length into tubes and adjust the concentration to 2 &#956;M for each strand.</li>
	</ol>
	</li>
	<li>Prepare MgCl2 solution of 300 nM by mixing 70 &#956;L of nuclease-free water and 30 &#956;L of the 1 &#956;M MgCl2 solution. Prepare a 5x Tris EDTA solution by mixing 95 &#956;L of nuclease-free water and 5 &#956;L of the 100x Tris EDTA solution.</li>
	<li>Add 2 &#956;L of staple DNA, 1.1 &#956;L of MgCl2 solution, 2 &#956;L of Tris EDTA solution, 7.6 &#956;L of nuclease-free water and 7.3 &#956;L of scaffold DNA of which the concentration is 110 nM to make 20 &#956;L of mixed stock. Set the final concentration of the scaffold DNA to 40 nM, staple DNA to 200 nM, MgCl2 to 16 mM and Tris EDTA solution to 0.5x.</li>
	<li>Rapidly heat the mixed stock solution in a thermal cycler to 80 &#176;C and cool to 60 &#176;C at a rate of 4 min per &#176;C and cool from 60 &#176;C to 4 &#176;C at a rate of 40 min per &#176;C.</li>
</ol>
4. Purification of the Structure
Note: Samples of all structures were purified before analysis. In this section, we describe the protocol of PEG purification, which is adapted from previous literature19. The sample can also be purified by gel electrophoresis as described in previous literature15,18.
<ol>
	<li>Prepare 5 M of NaCl and 100x Tris-EDTA.</li>
	<li>Prepare precipitation-buffer by mixing 150 &#956;L of PEG 8000, 500 &#956;L of 100x Tris EDTA and 101 &#956;L of 5 M NaCl and 249 &#956;L of nuclease-free water.</li>
	<li>Prepare target-buffer by mixing 5.5 &#956;L of 300 nM MgCl2 solution from Section 3.3, 10 &#956;L of 5x Tris EDTA solution from Section 3.3 and 84.5 &#956;L of nuclease-free water.</li>
	<li>Mix 20 &#956;L of the synthesized structure from Section 3 and 20 &#956;L of precipitation-buffer from Section 4.2. Then spin the mixed stock at 16000 x g at 4 &#176;C. Remove the supernatant and dissolve pellet in the target-buffer from Section 4.3.</li>
</ol>
5. Reconfiguration of the Accordion Rack from a &#8216;Free State&#8217; to a &#8216;Locked State&#8217;
<ol>
	<li>Synthesize the structure without DNA locks for the configuration experiment.</li>
	<li>Prepare DNA lock strands from Section 3.</li>
	<li>Add 2 &#956;L of DNA lock strands of the desired length into 20 &#956;L of the synthesized structure. The DNA lock strands&#8217; concentration is five times higher than the structure.</li>
	<li>Incubate the sample for 0, 10, 25, 50, or 100 minutes to see how fast reconfiguration occurs.
	<ol>
		<li>For the 100 minute incubation, incubate the sample at 50 &#176;C for 30 minutes and slowly cool down to 25 &#176;C at a rate of 0.33 &#176;C/minute.</li>
		<li>For the 50 minute incubation, incubate the sample at 50 &#176;C for 15 minutes and slowly cool down to 25 &#176;C at a rate of 0.66 &#176;C/minute.</li>
		<li>For the 25 minute incubation, incubate the sample at 50 &#176;C for 7.5 minutes and slowly cool down to 25 &#176;C at a rate of 1.32 &#176;C/minute.</li>
		<li>For the 10 minute incubation, incubate the sample at 50 &#176;C for 3 minutes and slowly cool down to 25 &#176;C at a rate of 3.3 &#176;C/minute.</li>
		<li>For the 0-minute incubation, store sample at 4 &#176;C right after DNA locks strands are added.</li>
	</ol>
	</li>
	<li>Right after the attaching step, rapidly cool down the sample to 4 &#176;C to prevent unwanted denaturation.</li>
</ol>
6. Reconfiguration of the Accordion Rack from a &#8216;Locked State&#8217; to a &#8216;Free State&#8217;
<ol>
	<li>Synthesize the structure with DNA locks of the desired length for the configuration experiment.</li>
	<li>Prepare reverse complementary strands from Section 3.</li>
	<li>Add 2 &#956;L of strands that are reverse complementary to the lock strands of the desired length into 20 &#956;L of the synthesized structure. The DNA lock strands&#8217; concentration is five times higher than the structure.</li>
	<li>Incubate the sample for 0, 12, 60, 120, 240 minutes to see how fast reconfiguration occurs.
	<ol>
		<li>For the 12, 60, 120, 240 minute incubation, rapidly heat the sample to 40 &#176;C and slowly cool down to 20 &#176;C for the time corresponding to each. Right after the detaching step, rapidly cool down the sample to 4 &#176;C to prevent unwanted denaturation.</li>
		<li>For the 0-minute incubation, store sample at 4 &#176;C right after reverse complementary strands are added.</li>
	</ol>
	</li>
</ol>
7. TEM Imaging
Note: TEM imaging protocol was adapted from previous literature18,20.
<ol>
	<li>Prepare 1.25 M NaOH solution by mixing 87.5 &#956;L of nuclease-free water and 12.5 &#956;L of 10 M NaOH solution.</li>
	<li>Add 1 &#956;L of 1.25 M NaOH solution to 50 &#956;L of the 2% uranyl formate solution.</li>
	<li>Vortex the solution for 3 minutes and centrifuge at max speed for 3 minutes. Deposit 3 &#956;L of the purified sample on the glow-discharged TEM grid for 3 minutes and rapidly wash out with filter paper.</li>
	<li>Deposit 7 &#956;L of prepared uranyl formate solution for 30 seconds and rapidly wash out with filter paper.</li>
	<li>Measure the length and angle of the accordion structure imaged by TEM.</li>
</ol>
8. FRET Analysis
<ol>
	<li>Use Atto 550 and Atto 647N dye, for which the F&#246;rster distance is 6.5 nm. Replace Staple 58 and Staple 117 in Table 1 with fluorescently labelled strands. Then synthesize the structure with fluorescently labelled strands by the method described in Section 3.</li>
	<li>Measure the concentration of the purified sample.</li>
	<li>Normalize the sample to 10 nM and load 50 &#956;L to the 384 microplates well.</li>
	<li>Excite the sample with donor and acceptor dyes at 550 nm and measure the fluorescence spectrum from 570 nm to 800 nm with a fluorometer.</li>
	<li>Measure the fluorescence spectrum of the donor-only sample in the same way.</li>
	<li>Excite the dyes of the sample at 650 nm and the fluorescence spectrum and measure from 670 nm to 800 nm. This is to measure the concentration of the acceptor.</li>
	<li>Obtain the standard deviations by repeating the same experiment with three samples, which are synthesized and purified separately.</li>
	<li>Calculate the FRET efficiency with the ratio A method as described by the equation below21. 
	<img alt="Equation" src="/files/ftp_upload/58364/58364eq1.jpg" /> 
	DA550: Acceptor peak fluorescence intensity of the sample with donor and acceptor at 550 nm excitation. 
	D550: Fluorescence intensity at the acceptor emission range of the donor-only sample at 550 nm excitation. 
	DA650: Acceptor peak fluorescence intensity of the sample with donor and acceptor at 650 nm excitation.</li>
</ol>

<ol>
	<li>Andersen, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+a+nanoscale+DNA+box+with+a+controllable+lid.">Self-assembly of a nanoscale DNA box with a controllable lid.</a> Nature. 459 (7243), 73-76 (2009).</li><li>Cha, T. -. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+principles+of+DNA+enzyme+based+walkers:+Translocation+kinetics+and+photo-regulation.">Design principles of DNA enzyme based walkers: Translocation kinetics and photo-regulation.</a> Journal of the American Chemical Society. 137 (29), 9429-9437 (2015).</li><li>Gerling, T., Wagenbauer, K. F., Neuner, A. M., Dietz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+DNA+devices+and+assemblies+formed+by+shape-complementary,+non-base+pairing+3D+components.">Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components.</a> Science. 347 (6229), 1446-1452 (2015).</li><li>Pinheiro, A. V., Han, D., Shih, W. M., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+opportunities+for+structural+DNA+nanotechnology.">Challenges and opportunities for structural DNA nanotechnology.</a> Nature nanotechnology. 6 (12), 763-772 (2011).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+speed+limit+of+toehold+exchange+with+a+cartwheeling+DNA+acrobat.">Exploring the speed limit of toehold exchange with a cartwheeling DNA acrobat.</a> Nature Nanotechnology. 1, (2018).</li><li>Krishnan, Y., Simmel, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+based+molecular+devices.">Nucleic acid based molecular devices.</a> Angewandte Chemie International Edition. 50 (14), 3124-3156 (2011).</li><li>Liu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+tweezer-actuated+enzyme+nanoreactor.">A DNA tweezer-actuated enzyme nanoreactor.</a> Nature communications. 4, 2127 (2013).</li><li>Douglas, S. M., Bachelet, I., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+logic-gated+nanorobot+for+targeted+transport+of+molecular+payloads.">A logic-gated nanorobot for targeted transport of molecular payloads.</a> Science. 335 (6070), 831-834 (2012).</li><li>Kuzyk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconfigurable+3D+plasmonic+metamolecules.">Reconfigurable 3D plasmonic metamolecules.</a> Nature Materials. 13 (9), 862-866 (2014).</li><li>Zhou, C., Duan, X., Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plasmonic+nanorod+that+walks+on+DNA+origami.">A plasmonic nanorod that walks on DNA origami.</a> Nature communications. 6, 8102 (2015).</li><li>Choi, Y., Choi, H., Lee, A. C., Lee, H., Kwon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Reconfigurable+DNA+Accordion+Rack.">A Reconfigurable DNA Accordion Rack.</a> Angewandte Chemie International Edition. 57 (11), 2811-2815 (2018).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Mechanical+Properties+of+DNA+Origami+Tiles+and+Controlling+the+Kinetics+of+their+Folding+and+Unfolding+Reconfiguration.">Understanding the Mechanical Properties of DNA Origami Tiles and Controlling the Kinetics of their Folding and Unfolding Reconfiguration.</a> Journal of the American Chemical Society. 136 (19), 6995-7005 (2014).</li><li>Han, D., Pal, S., Liu, Y., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+and+cutting+DNA+into+reconfigurable+topological+nanostructures.">Folding and cutting DNA into reconfigurable topological nanostructures.</a> Nature Nanotechnology. 5 (10), 712-717 (2010).</li><li>Douglas, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+prototyping+of+3D+DNA-origami+shapes+with+caDNAno.">Rapid prototyping of 3D DNA-origami shapes with caDNAno.</a> Nucleic Acids Research. 37 (15), 5001-5006 (2009).</li><li>Castro, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+to+scaffolded+DNA+origami.">A primer to scaffolded DNA origami.</a> Nature methods. 8 (3), 221-229 (2011).</li><li>Ouldridge, T. E., Louis, A. A., Doye, J. P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Nanotweezers+Studied+with+a+Coarse-Grained+Model+of+DNA.">DNA Nanotweezers Studied with a Coarse-Grained Model of DNA.</a> Physical Review Letters. 104 (17), 178101 (2010).</li><li>Snodin, B. E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Simulation+of+the+Self-Assembly+of+a+Small+DNA+Origami.">Direct Simulation of the Self-Assembly of a Small DNA Origami.</a> ACS Nano. 10 (2), 1724-1737 (2016).</li><li>Amir, Y., Abu-Horowitz, A., Bachelet, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+and+Characterization+of+a+Bio-responsive+Robot+from+DNA+Origami.">Folding and Characterization of a Bio-responsive Robot from DNA Origami.</a> Journal of Visualized Experiments. (106), e51272 (2015).</li><li>Stahl, E., Martin, T. G., Praetorius, F., Dietz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+and+Scalable+Preparation+of+Pure+and+Dense+DNA+Origami+Solutions.">Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions.</a> Angewandte Chemie International Edition. 53 (47), 12735-12740 (2014).</li><li>Wei, B., Vhudzijena, M. K., Robaszewski, J., Yin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+Complex+Two-dimensional+Shapes+from+Single-stranded+DNA+Tiles.">Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles.</a> Journal of Visualized Experiments. (99), e52486 (2015).</li><li>Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+and+nucleic+acids.">Fluorescence resonance energy transfer and nucleic acids.</a> Methods in enzymology. 211, 353-388 (1992).</li><li>Kopperger, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-assembled+nanoscale+robotic+arm+controlled+by+electric+fields.">A self-assembled nanoscale robotic arm controlled by electric fields.</a> Science. 359 (6373), 296-301 (2018).</li><li>Lauback, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+magnetic+actuation+of+DNA+nanodevices+via+modular+integration+with+stiff+micro-levers.">Real-time magnetic actuation of DNA nanodevices via modular integration with stiff micro-levers.</a> Nature Communications. 9 (1), 1446 (2018).</li></ol>

The authors have nothing to disclose

This protocol introduces the entire process from design, simulation, synthesis, and analysis of the basic 2D DNA accordion rack. The modified design and simulation rules have been described because the design rule differs from that of standard DNA origami, in that the DNA accordion rack has additional nucleotides at the crossovers for flexibility14,15. From this, we expect that the protocol can accelerate various researches using DNA accordion racks. In addition, the described protocol can also be applied to other research using DNA nanostructure rather than standard DNA origami.
For structures other than the basic 2D structure demonstrated in the original research paper, the design can be done with modifications of the described protocol. In brief, a boxing-glove-spring structure is designed by changing the beam number and length from the basic model. 3D nanotubular structures can also be created by connecting both ends of a 2D accordion rack structure. However, for simulating the 3D structures, the initial position of the beams should be considered in 3D space and the initial state of the beams is supposed to be bent. Therefore, more computational transformation is needed. Design and simulation of various 2D and 3D DNA accordion rack structures will be conducted by developing the computer-aided program in future research.
Speed and repeatability of actuation are also important factors in the field of dynamic DNA nanotechnology. Recently dynamic DNA structures that respond to external electrical or magnetic field are proposed and operated with high speed22,23. While the DNA accordion rack has enabled collective actuation of multiple DNA beams, the reconfiguration speed was not significantly improved since actuation is based on DNA strand displacement. For further applications, response to external stimuli should be improved by optimizing DNA lock design or using another external stimuli.
As an application study for the reconfigurable accordion structure, various functional materials such as drug molecules, nanoparticles, or proteins can be attached to the structure. For this, the molecules can be connected to the staple that exists at the intended position. Overall, a variety of application studies can be conducted through this protocol and its modifications.

Mechanical systems based on DNA nanostructures or DNA nanomachines1,2,3,4,5 are unique because they produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, according to various biomolecular stimuli2,3,6. By attaching functional materials on these structures and controlling their positions, these structures can be applied to various areas. For example, DNA nanomachines have been proposed for a molecular reactor7, drug delivery8, and nanoplasmonic systems9,10.
Previously, we introduced the reconfigurable DNA accordion rack, which can manipulate a 2D or 3D nanoscale network of elements11 (Figure 1A). Unlike other DNA nanomachines that only control a few elements, the platform can collectively manipulate periodically arrayed 2D or 3D elements into various stages. We anticipate that a programmable chemical and biological reaction network or a molecular computing system can be built from our system, by increasing the number of controllable elements. The DNA accordion rack is a structure, in which the network of multiple DNA beams is connected to joints composed of single-stranded DNA (Figure 1B). The accordion rack generated by the DNA beams is reconfigured by the DNA locks, which hybridize to the sticky part of beams and change the angle between the beams according to the length of the bridging part of the locks (locked state). In addition, multi-step reconfiguration is demonstrated by adding new locks after formation of the free state by detaching DNA locks through toehold-based strand displacement12,13.
In this protocol, we describe the entire design and synthesis process of the reconfigurable DNA accordion rack. The protocol includes design, simulation, wet-lab experiments, and analysis for the synthesis of the DNA accordion rack of 6 by 6 meshes and a reconfiguration of these. The structure covered in the protocol is the basic model of the previous research11 and is 65 nm by 65 nm in size, consisting of 14 beams. In terms of the design and simulation, the structural design of the accordion rack is different from conventional DNA origami14,15 (i.e., tightly packed). Therefore, the design rule and molecular simulation have been modified from traditional methods. To demonstrate, we show the design technique using the modified approach of caDNAno14 and the simulation of the accordion rack using oxDNA16,17 with additional scripts. Finally, both protocols of TEM and FRET for analysis of configured accordion rack structures are described.

<table>
	<tbody>
		<tr>
			<td>M13mp18 Single-stranded DNA</td>
			<td>NEB</td>
			<td>N4040s</td>
			<td></td>
		</tr>
		<tr>
			<td>1M MgCl2 Solution</td>
			<td>Biosesang</td>
			<td>M2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-EDTA buffer</td>
			<td>Biosesang</td>
			<td>T2142</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water</td>
			<td>Qiagen</td>
			<td>129114</td>
			<td></td>
		</tr>
		<tr>
			<td>5M Sodium Chloride solution</td>
			<td>Biosesang</td>
			<td>s2007</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>Sigma Aldrich</td>
			<td>1546605</td>
			<td></td>
		</tr>
		<tr>
			<td>10N NaOH</td>
			<td>Biosesang</td>
			<td>S2038</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl formate</td>
			<td>Thomas Science</td>
			<td>C993L42</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal cycler C1000</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodropic 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEM (LIBRA 120)&#160;&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer Enspire 2300</td>
			<td>Perkin-Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Labogene</td>
			<td>LZ-1580</td>
			<td></td>
		</tr>
	</tbody>
</table>

design and synthesis of a reconfigurable dna accordion rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

Watch this Scientific Journal Video about Design and Synthesis of a Reconfigurable DNA Accordion Rack at JoVE.com

Design and Synthesis of a Reconfigurable DNA Accordion Rack

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

design-and-synthesis-of-a-reconfigurable-dna-accordion-rack

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.0K Views.  Seoul National University. We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

Video: Design and Synthesis of a Reconfigurable DNA Accordion Rack

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Reconfigurable Microfluidic Channel with Pin-discretized Sidewalls

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or reactions. A microfluidic channel that deforms even after fabrication while retaining the channel shape enables high spatiotemporal reconfigurability. This reconfigurability is required in such key microfluidic functions that are difficult to achieve in existing "reconfigurable" or "integrated" microfluidic systems. We describe a method for the fabrication of a microfluidic channel with a deformable sidewall consisting of a laterally aligned array of the ends of rectangular pins. Actuating the pins in their longitudinal directions changes the pins' end positions, and thus, the shape of discretized channel sidewalls.Pin gaps can cause unwanted leakage or adhesion to adjacent pins caused by meniscus forces. To close the pin gaps, we have introduced hydrocarbon-fluoropolymer suspension-based gap filler accompanied by an elastomeric barrier. This reconfigurable microfluidic device can generate strong temporal in-channel displacement flow, or can stop the flow in any region of the channel. This feature will facilitate, on demand, the handling of cells, viscous liquids, gas bubbles, and non-fluids, even if their existence or behavior is unknown at the time of fabrication.

A microfluidic channel with deformable sidewalls offers flow control, particle handling, channel dimension customization and other reconfigurations while in use. We describe a method for fabricating a microfluidic channel with sidewalls made of an array of pins that allows their shape to change.

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...

Design of a Biocompatible Drug-Eluting Tracheal Stent in Mice with Laryngotracheal Stenosis

Laryngotracheal stenosis (LTS) is a pathologic narrowing of the subglottis and trachea leading to extrathoracic obstruction and significant shortness of breath. LTS results from mucosal injury from a foreign body in the trachea, leading to tissue damage and a local inflammatory response that goes awry, leading to the deposition of pathologic scar tissue. Treatment for LTS is surgical due to the lack of effective medical therapies. The purpose of this method is to construct a biocompatible stent that can be miniaturized to place into mice with LTS. We demonstrated that a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) construct had optimal biomechanical strength, was biocompatible, practicable for an in vivo placement stent, and capable of eluting drug. This method provides a drug delivery system for testing various immunomodulatory agents to locally inhibit inflammation and reduce airway fibrosis. Manufacturing the stents takes 28&#8722;30 h and can be reproduced easily, allowing for experiments with large cohorts. Here we incorporated the drug rapamycin within the stent to test its effectiveness in reducing fibrosis and collagen deposition. Results revealed that PLLA-PCL tents showed reliable rapamycin release, were mechanically stable in physiological conditions, and were biocompatible, inducing little inflammatory response in the trachea. Further, the rapamycin-eluting PLLA-PCL stents reduced scar formation in the trachea in vivo.

Laryngotracheal stenosis results from pathologic scar deposition that critically narrows the tracheal airway and lacks effective medical therapies. Using a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) stent as a local drug delivery system, potential therapies aimed at decreasing scar proliferation in the trachea can be studied.

Laryngotracheal stenosis (LTS) is a pathologic narrowing of the subglottis and trachea leading to extrathoracic obstruction and significant shortness of ...