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>

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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 MgCl&lt;sub&gt;2&lt;/sub&gt; 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)&amp;nbsp;&amp;nbsp;</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

Universidad San Sebastian

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

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Chemistry

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

Genetics

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...

DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA nanofabrication technique is called DNA origami, and it allows high-throughput synthesis of accurate and highly versatile structures with nanometer-level precision. Here, it is shown how the spatial information of DNA origami can be transferred to metallic nanostructures by combining the bottom-up DNA origami with the conventionally used top-down lithography approaches. This allows fabrication of billions of tiny nanostructures in one step onto selected substrates. The method is demonstrated using bowtie DNA origami to create metallic bowtie-shaped antenna structures on silicon nitride or sapphire substrates. The method relies on the selective growth of a silicon oxide layer on top of the origami deposition substrate, thus resulting in a patterning mask for following lithographic steps. These nanostructure-equipped surfaces can be further used as molecular sensors (e.g., surface-enhanced Raman spectroscopy (SERS)) and in various other optical applications at the visible wavelength range owing to the small feature sizes (sub-10 nm). The technique can be extended to other materials through methodological modifications; therefore, the resulting optically active surfaces may find use in development of metamaterials and metasurfaces.

Here, we describe a protocol to create discrete and accurate inorganic nanostructures on substrates using DNA origami shapes as guiding templates. The method is demonstrated by creating plasmonic gold bowtie-shaped antennas on a transparent substrate (sapphire).

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA ...

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; ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...