Name: design and synthesis of a reconfigurable dna accordion rack
Uploaded: 2018-08-15T15:00:00
Description: Watch this Scientific Journal Video about Design and Synthesis of a Reconfigurable DNA Accordion Rack at JoVE.com

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 (<strong cl...

<ol>
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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,...

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.

The designed 6 by 6 DNA accordion rack is simulated from the oxDNA16,17 and the results are shown in Figure 6. From the simulation result, it was confirmed that the intended structure is formed without distortion of the structure.
The TEM images in Figure 7 are images of configured structures with a lock length ...

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 ...

This method can help answer key questions in the nano molecules field, such as DNA nano technology, nano molecular circuits, and nano medicine. The main advantage of this technique is that it dramatically increases the category of controlling periodically irate elements in both two and three dimensions. To begin, download and install Cadnano software to design a DNA accordion rack.Open Cadnano and click the square tool to add a new part with a square lattice. Click the pencil tool and draw each beam on the right edit panel of Cadnano. Break beams every 32 base pairs for joints between adjacent beams.Place staple crossovers in the same position as the joints. Use the insert tool and the pencil tool in Cadnano to let the joints have additional single stranded crossovers. Now, click the break tool.Break the strands where staple strands are circular or longer than 60 base pairs. To design the DNA lockstrands, click the break tool. Break eight base pairs of a staple DNA region to make a sticky part and delete eight base pairs of a staple DNA region.There are 18 sticky parts in the six by six accordion rack. Place sequences that are reverse complimentary to the sticky parts at both ends of lockstrands and connect them by a bridging region, which consists of poly T strands of the desired length. For the reconfiguration, add eight base pairs of toehold sequences at the end of the DNA locks for strand displacement.Design strands that are reverse complimentary to the DNA locks for the reconfiguration experiment. Generate a topology file and configuration file using OxDNA as described in the text protocol. 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 time step, box size, and energy. Orientation information, such as position vector, backbone-base vector, normal vector, velocity and angular velocity of nucleotides is also included. Change the information in the topology and configuration file from Cadnano to make them reflect the real structural information of the accordion rack.Then, run the molecular dynamics program as described in the text protocol. To visualize the structures, run the Cogli program with the topology and configuration files from the OxDNA simulation. Hide the box by pressing B.Purchase the design DNA staples and prepare them as described in the text protocol.Combine staple DNA, magnesium chloride solution, tris EDTA solution, nuclease free water, and scaffold DNA to make 20 micro liters of mixed stock with the final concentrations listed in the text protocol. Now, rapidly heat the mixed stock solution in a thermal cycler to 80 degrees Celsius. Cool the solution to 60 degrees Celsius, at a rate of four minutes per degree Celsius.Then, cool from 60 degrees Celsius to four degrees Celsius at a rate of 40 minutes per degree Celsius. To purify the structure, mix 20 micro liters of the synthesized structure in 20 micro liters of precipitation buffer. Then, spin the mixed stock at 16, 000 times G and four degrees celsius.Remove the super natant and dissolve pellet in the target buffer. To reconfigure the accordion rack from a free state to a locked state, first add two micro liters of DNA lockstrands of the desired length into 20 micro liters of the synthesized structure. Incubate the sample for 100 minutes at 50 degrees Celsius for 30 minutes and slowly cool down to 25 degrees Celsius at a rate of 0.33 degrees Celsius per minute.To reconfigure the accordion rack from a locked state to a free state, add two micro liters of strands that are reverse complimentary to the locked strands of the desired length into 20 microliters of synthesized structure. Incubate the sample for 12, 60, 120, and 240 minutes by rapidly heating the sample to 40 degrees Celsius and slowly cooling down to 20 degrees Celsius for the time corresponding to each. Right after the detaching step, rapidly cool down the sample to four degrees Celsius to prevent unwanted denaturation.To perform FRET analysis, synthesize the structure as before, expect with fluorescently labeled strands. Measure the concentration of the purified sample. After normalizing the sample to 10 nano molar, load 50 micro liters to a well in a 384 well micro plate.Measure the fluorescence of the sample with donor and acceptor dyes, as well as the donor only sample by exciting at 550 nano meters and measuring the fluorescent spectrum from 570 nano meters to 800 nano meters with a fluorometer. To measure the concentration of the acceptor, excite the dyes of the sample at 650 nano meters and measure the fluorescent spectrum from 670 nano meters to 800 nanometers. Then, calculate the FRET efficiency as described in the text protocol.A simulation result from OxDNA is visualized here by Cogli. The accordion rack is shown with DNA locks, of which the lengths are two, eight, 13, and 20 base pairs. The angle of the accordion rack, comprised of six by six meshes, was controlled by the length of the DNA locks.By adding DNA locks with lengths of two, eight, 13, and 20 base pairs to the 18 locking sights of the accordion rack, the angle is configured as seen in the representative transmission electron microscopy, or TEM images. The dye pair is attached to the structure for FRET efficiency analysis. Shown here are representative TEM images of configured six by six DNA accordion racks with different DNA lock lengths.After its development, this technique paved the way for researchers in the field of DNA nano technology to explore interactions of different molecules in a collectively controlled system.

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