The authors wish to thank S. Douglas for extremely valuable discussions and advice, and all the members of the Bachelet lab for helpful discussions and work. This work is supported by grants from the Faculty of Life Sciences and Institute of Nanotechnology &#38; Advanced Materials at Bar-Ilan University.

1. Preparation of Staples Pool Mixture
<ol>
	<li>Order lyophilized DNA nanorobot staples on 96-well plates as listed in Table 1 (see Materials) and normalize to 10 nmol. For a detailed description of the design and architecture of the DNA nanorobot see Ben-Ishay et al.14 and Douglas et al.15).</li>
	<li>Reconstitute each staple well with DNase/RNase-free ultrapure to a concentration of 100 &#181;M. For staples normalized to 10 nmol, reconstitute with 100 &#181;l of ultrapure water.</li>
	<li>Pool together 20 &#181;l of each staple using a multichannel pipettor and a sterile 55 ml solution basin. 
	Note: Since the nanorobot shape is comprised of 254 staple strands (including Core, Edges, Handles and Guides sequences, Table 1), the concentration of each of the staples in the staples pool is 394 nM. Removing staples from the staple pool, or adding some at different volumes, may reduce the yield considerably, or otherwise result in unfolded aggregates.</li>
</ol>
2. Preparation of Fabrication Reaction Mixture
<ol>
	<li>For a standard reaction mixture use 40 &#181;l of M13mp18 viral genome circular single strand DNA, corresponding to 4 pmol of scaffold DNA (100 nM stock solution, see Materials). Final concentration of scaffold in the fabrication mixture is 20 nM, final volume 200 &#181;l.
	<ol>
		<li>Adjust the amount of scaffold DNA according to specific needs; however, keep the final concentration of scaffold DNA in the reaction mixture at a constant 20 nM.</li>
	</ol>
	</li>
	<li>Add staples to reach a scaffold to staples ratio of 1 to 10, respectively. For a 20 nM scaffold DNA concentration, each of the 254 staples&#8217; final concentration is 200 nM. For a 200 &#181;l final reaction volume add 102 &#181;l of the staples pool mixture (section 1.2).
	<ol>
		<li>Add specific Gate sequences separately to the folding reaction mixture at this time. These oligos are ordered separately and require HPLC purification. Ensure that Gate oligos are present in a 1:10 scaffold to Gate sequence ratio, i.e. 200 nM of each oligo for a 20 nM scaffold concentration. For a 200 &#181;l folding reaction volume, add 0.4 &#181;l for each of the four Gate oligo at a 100 &#181;M stock concentration.</li>
	</ol>
	</li>
	<li>Add 10x TAE stock buffer to reach a final concentration of 1x TAE (40 mM Tris-Acetate, 1 mM EDTA). For a 200 &#181;l folding reaction volume, add 20 &#181;l of 10x TAE.</li>
	<li>Add 1 M MgCl2 to a final concentration of 10 mM. For a 200 &#181;l folding reaction volume, add 2 &#181;l of 1 M MgCl2.</li>
	<li>Add 36 &#181;l of DNase/RNase free ultrapure water to a reach a final volume of 200 &#181;l.</li>
	<li>Vortex and aliquot 100 &#181;l samples into PCR vials. 
	Note: Consult thermal cycler specification regarding the maximum reaction volumes to be used. Reducing the volume to meet these limits will not reduce the yields obtained. Reaction volumes above the maximum specified will jeopardize the yields.</li>
</ol>
3. Temperature Annealing Ramp of Fabrication Reaction
<ol>
	<li>Program thermal cycler as followed:
	<ol>
		<li>Ramp 85 &#176;C to 60 &#176;C at a rate of 5 min/&#176;C.</li>
		<li>Ramp 60 &#176;C to 4 &#176;C at a rate of 75 min/&#176;C.</li>
		<li>Hold at 4 &#176;C indefinitely.</li>
	</ol>
	</li>
	<li>After fabrication has ended store samples at -20 &#176;C.</li>
</ol>
4. Removal of Excess Staples
<ol>
	<li>Add 100 &#181;l of folding reaction mixture to a 0.5 ml centrifugal&#160;filter&#160;with a MWCO&#160;of 100 kDa. Save a 10 &#181;l sample of nanorobots pre-purification for later analysis.</li>
	<li>Centrifuge for 10 min at 9,600 x g.</li>
	<li>Add 400 &#181;l of folding buffer (1x TAE, 10 mM MgCl2).</li>
	<li>Repeat steps 4.2 and 4.3 twice more.</li>
	<li>Centrifuge for 5 min at 9,600 x g.</li>
	<li>Recover the concentrate by placing the filter upside down in a clean microcentrifuge tube and spin for 1 min at 9,600 x g. Final volumes may vary depending on the initial volume of fabrication reaction mixture that was put into the filter. Typically a 25-60 &#181;l final volume of concentrated nanorobot sample is obtained.</li>
	<li>Measure concentration of DNA in the samples via spectrophotometer at 260 nm. Use molecular weight of 5.3 &#181;g/pmol when calculating molar concentrations of nanorobot samples. 
	Note: The molar extinction coefficient for dsDNA, 50 &#181;g/OD260, is sufficient for most applications. 48 &#181;g/OD260 takes into account the poly thymine ssDNA stretches at the Edges staples.</li>
</ol>
5. Agarose Gel Electrophoresis Analysis of Folded Nanorobots
<ol>
	<li>Preparation of 0.5x TBE (45 mM Tris-Borate, 1 mM EDTA), 2% agarose gel supplemented with 10 mM MgCl2 (adapted from Ernesto Castro et al.21):
	<ol>
		<li>Prepare 0.5x TBE buffer by diluting 6.25 ml of 10x TBE stock buffer in 118.75 ml of ddH2O.</li>
		<li>Dissolve 2.5 g of agarose in 125 ml of 0.5x TBE buffer.</li>
		<li>Boil in microwave until the agarose is completely dissolved.</li>
		<li>Add 1.25 ml of 1 M MgCl2 to final concentration of 10 mM.</li>
		<li>Add 7 &#181;l of 10 mg/ml ethidium bromide.</li>
		<li>Wait for solution to slightly cool and fill the gel tray before the agarose gel solidifies. Install desired comb immediately.</li>
		<li>Prepare 1 L 0.5x TBE running buffer by adding 50 ml of 10x TBE stock buffer and 10 ml of 1 M MgCl2 to 940 ml of ddH2O.</li>
		<li>Once gel is solid add running buffer to the electrophoresis device and put device in an ice bath.</li>
	</ol>
	</li>
	<li>Load 1 &#181;g total DNA of the nanorobots pre-purification (step 3.2), and post purification (step 4.7), alongside a scaffold DNA sample and a 1 kb DNA marker, onto the gel. An example is given in Figure 2. Actual volumes of samples depend on the concentrations obtained after purification. Each samples is loaded with loading buffer at a 1:6 final volume ratio.</li>
	<li>Set power source to 80 V and run gel for 3 hr. Run the gel in a bath filled with ice and water. Nanorobots will unfold and appear as a smear if the agarose gel heats up during electrophoresis. During electrophoresis add additional ice to keep device from heating.</li>
	<li>View gel on a UV table (Figure 2).</li>
</ol>
6. Negative Stain of Nanorobot with Uranyl-formate

<ol>
	<li>Preparation of 2% uranyl-formate stock solution (adapted from Castro et al.23):
	<ol>
		<li>Weigh 100 mg of uranyl-formate powder into a 15 ml tube.</li>
		<li>Boil DNase/RNase free ultrapure water for 3 min to de-oxygenate.</li>
		<li>Add 5 ml of deoxygenated hot water (~60 &#176;C) into the 15 ml tube containing the uranyl-formate powder (from previous step). Tightly close lid, wrap in aluminum foil and vortex rigorously for 10 min (fasten tube to a vortex). Solution should appear turbid with a yellow color.</li>
		<li>Filter solution through a 0.2 &#181;m syringe &#64257;lter. Solution should become clear.</li>
		<li>Aliquot 200 &#181;l of the solution into microcentrifuge tubes.</li>
		<li>Centrifuge at max speed for 5 min in a table top centrifuge to pool samples at the bottom of the tube.</li>
	</ol>
	</li>
	<li>Loading of sample to grid and negative staining:
	<ol>
		<li>Defrost a 200 &#181;l aliquot of 2% uranyl-formate solution (prepared in section 6.1). Add 10 &#181;l of 0.5 M NaOH solution and vortex rigorously for 3 min. Centrifuge at max speed for 5 minutes using a table top centrifuge.</li>
		<li>Meanwhile, glow-discharge grid using room air at 0.2 mbar and 25 mA for 30 sec.</li>
		<li>Add 15 &#181;l of 2 nM nanorobot sample after purification (section 4.7) onto topside of grid (held with forceps) and let absorb for 1 min. Use &#64257;lter paper to drain the excess liquid by placing the grid on top of the filter paper. Do not touch the grid surface.</li>
		<li>Add 10 &#181;l of the 2% uranyl-formate (from step 6.2.1) onto the topside of grid (held with forceps).</li>
		<li>Quickly use &#64257;lter paper to drain the excess liquid by placing the grid on top of the filter paper. Do not touch the grid surface.</li>
		<li>Repeat steps 6.2.4 and 6.2.5.</li>
		<li>Add 10 &#181;l of the 2% uranyl-formate (from step 6.2.1) onto the topside of grid (held with forceps). Let staining solution absorb for 30 sec.</li>
		<li>Use &#64257;lter paper to drain the excess liquid by placing the grid on top of the filter paper. Do not touch the grid surface.</li>
		<li>Let the grid dry completely for at least 30 min, face up on a filter paper, before injecting into the TEM.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Watson, J. D., Crick, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetical+implications+of+the+structure+of+deoxyribonucleic+acid.">Genetical implications of the structure of deoxyribonucleic acid.</a> Nature. 171, 964-967 (1953).</li><li>Kosuri, S., 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=Large-Scale+de+novo.+DNA+synthesis:+technologies+and+applications.">Large-Scale de novo. DNA synthesis: technologies and applications.</a> Nature Meth. 11 (5), 499-507 (2014).</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 Nanotech. 6 (12), 763-772 (2011).</li><li>Seeman, N. 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+junctions+and+lattices.">Nucleic acid junctions and lattices.</a> J Theor Biol. 99 (2), 237-247 (1982).</li><li>Chen, J. H., Seeman, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+from+DNA+of+a+molecule+with+the+connectivity+of+a+cube.">Synthesis from DNA of a molecule with the connectivity of a cube.</a> Nature. 350 (6319), 631-633 (1991).</li><li>Wei, B., Dai, M., 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=Complex+shapes+self-assembled+from+single-stranded+DNA+tiles.">Complex shapes self-assembled from single-stranded DNA tiles.</a> Nature. 485 (7400), 623-626 (2012).</li><li>He, Y., 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=Hierarchical+self-assembly+of+DNA+into+symmetric+supramolecular+polyhedra.">Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra.</a> Nature. 452 (7184), 198-201 (2008).</li><li>Yin, P., Hariadi, R. F., Sahu, S., Choi, H. M. T., Park, S. H., Labean, T. H., Reif, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+DNA+tube+circumferences.">Programming DNA tube circumferences.</a> Science. 321 (5890), 824-826 (2008).</li><li>Rothemund, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+DNA+to+create+nanoscale+shapes+and+patterns.">Folding DNA to create nanoscale shapes and patterns.</a> Nature. 440 (7082), 297-302 (2006).</li><li>Dietz, H., Douglas, S. M., Shih, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+DNA+into+twisted+and+curved+nanoscale+shapes.">Folding DNA into twisted and curved nanoscale shapes.</a> Science. 325 (5941), 725-730 (2009).</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=Self-assembly+of+DNA+into+nanoscale+three-dimensional+shapes.">Self-assembly of DNA into nanoscale three-dimensional shapes.</a> Nature. 459 (7245), 414-418 (2009).</li><li>Zhang, F., Nangreave, J., 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=Structural+DNA+nanotechnology:+state+of+the+art+and+future+perspective.">Structural DNA nanotechnology: state of the art and future perspective.</a> J Am Chem Soc. 136 (32), 11198-11211 (2014).</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=Prototyping+of+3D+DNA-origami+shapes+with+caDNAno.">Prototyping of 3D DNA-origami shapes with caDNAno.</a> Nucleic Acids Res. 37 (15), 5001-5006 (2009).</li><li>Ben-Ishay, E., 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=Designing+a+bio-responsive+robot+from+DNA+origami.">Designing a bio-responsive robot from DNA origami.</a> J Vis Exp. (77), e50268 (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>Tan, W., Donovan, M. J., Jiang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers+from+cell-based+selection+for+bioanalytical+applications.">Aptamers from cell-based selection for bioanalytical applications.</a> Chem Rev. 113 (4), 2842-2862 (2013).</li><li>Xiang, D., 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=Nucleic+Acid+Aptamer-Guided+Cancer+Therapeutics+and+Diagnostics:+The+Next+Generation+of+Cancer+Medicine.">Nucleic Acid Aptamer-Guided Cancer Therapeutics and Diagnostics: The Next Generation of Cancer Medicine.</a> Theranostics. 5 (1), 23-42 (2015).</li><li>Zhang, D. Y., Seelig, G. <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+nanotechnology+using+strand-displacement+reactions.">Dynamic DNA nanotechnology using strand-displacement reactions.</a> Nat Chem. 3 (2), 103-113 (2011).</li><li>Tang, Z., Parekh, P., Turner, P., Moyer, R. W., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+aptamers+for+recognition+of+virus-infected+cells.">Generating aptamers for recognition of virus-infected cells.</a> Clin Chem. 55 (4), 813-822 (2009).</li><li>Sefah, K., Shangguan, D., Xiong, X., O'Donoghue, M. B., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+DNA+aptamers+using+Cell-SELEX.">Development of DNA aptamers using Cell-SELEX.</a> Nature Prot. 5 (6), 1169-1185 (2010).</li><li>McKeague, M., DeRosa, M. C. <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+Small+Molecule+Aptamer+Development.">Challenges and Opportunities for Small Molecule Aptamer Development.</a> J Nucleic Acids. 2012, (2012).</li><li>Amir, Y., 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=Universal+computing+by+DNA+origami+robots+in+a+living+animal.">Universal computing by DNA origami robots in a living animal.</a> Nature Nanotech. 9 (5), 353-357 (2014).</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 Meth. 8 (3), 221-229 (2011).</li><li>Sobczak, J. P., Martin, T. G., Gerling, T., 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=Rapid+folding+of+DNA+into+nanoscale+shapes+at+constant+temperature.">Rapid folding of DNA into nanoscale shapes at constant temperature.</a> Science. 338 (6113), 1458-1461 (2012).</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> Angew Chem Int Ed Engl. 53 (47), 12735-12740 (2014).</li><li>Lin, C., Perrault, S. D., Kwak, M., Graf, F., Shih, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+DNA-origami+nanostructures+by+rate-zonal+centrifugation.">Purification of DNA-origami nanostructures by rate-zonal centrifugation.</a> Nucleic Acids Res. 41 (2), (2012).</li><li>Bai, X. C., Martin, T. G., Scheres, S. H., 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=Cryo-EM+structure+of+a+3D+DNA-origami+object.">Cryo-EM structure of a 3D DNA-origami object.</a> Proc Natl Acad Sci U S A. 109 (49), 20012-20017 (2012).</li></ol>

The authors have nothing to disclose.

We described the fabrication, purification, and visualization of the DNA nanorobot. Following fabrication of the hexagonal chassis of the device, the function of the nanorobot is programmed with the simple introduction of specific cargo and sensing strands to the robot which readily find their designated position due to hydrogen-bonding complementarity with available single-strand docking sites14,15,22.
The fabrication protocol described uses a slow annealing ramp, which is generall...

The uses for nucleic acids nanotechnology are astounding. The tractability of the Watson-Crick base pairing as well as the ease and relative low-cost of large-scale synthesis of custom-made oligos2 has generated an explosion of applications3 and research in the field of DNA nanotechnology. Structural DNA nanotechnology, based on the immobile Seeman junction4,5 as a fundamental building block makes use of DNA as a self-assembling elementary unit for the construction of arbitrary shapes6-8.
The recent development of the scaffolded DNA origami9 technique allows for the construction of complex 2D/3D nano-architectures10-12 with sub-nanometer precision and is an efficient route for building new functional objects with increasing complexity and astonishing diversity. The construction process is based upon a long scaffold single stranded DNA, usually derived from a viral genome, which can be folded through the hybridization of hundreds of short single strand DNA oligos termed staples. The high structural resolution obtained by this technique is the direct result of the natural dimensions of the DNA double helix, while the reproducibility of fabrication is the result of tailoring the short single-strand staple sequences to facilitate the maximum hydrogen-bonding complementarity achievable. With the use of a slow temperature annealing ramp the designed lowest-energy, thermodynamically preferred nanostructure is reached in high yields and fidelity. The easy implementation of junction design rules in a computer code enabled the development of CAD tools, such as caDNAno13, that extremely simplify the task of designing large, complex structures containing hundreds of connected junctions.
Previously we described the design of a DNA nanorobot with the aid of the caDNAno tool14,15. Here we depict the fabrication and visualization, via transmission electron microscopy (TEM), of the nanorobot, a 3D hollow hexagonal nanodevice, with dimensions of 35 x 35 x 50 nm3, designed to undergo a major conformational change in response to a predetermined stimuli and present specific cargo, such as proteins or nucleic acid oligos, sequestered inside. While 12 loading stations are available inside the hollow chassis, the actual number of bound cargo differs with cargo size. Cargo molecules range from small DNA molecules to enzymes, antibodies and 5-10 nm gold nanoparticles. Cargocan either be uniform or heterogeneous, such that each nanorobot contains a mixture of different molecules. Sensing is achieved via two double helical locking gates design to sense proteins, nucleic acids or other chemicals, based either on aptasensor16,17 or DNA strand displacement18 technologies. Recent developments in aptamer selection protocols19-21 enable the design of nanorobots responding to an ever increasing range of molecules and cell types.
Earlier work showed a nanorobot carrying a specific antibody, which upon binding to its antigen can relay either an inhibitory or a prolific signal to the inside of specific cell types in a mixed cell population15. An exciting feature of these nanodevices is their ability to perform even more complex tasks and logic control with the introduction of different nanorobot subtypes in a single population. Recently we demonstrated specific subtypes of nanorobots performing as either positive or negative regulators, controlling an effector population containing an active cargo molecule22.
The protocol presented here describes the fabrication, purification and imaging of a nanorobot gated with aptamer sensor sequences which bind selectively to PDGF to facilitate the opening of the nanorobot15,22. The fabrication process described is similar to the nanorobot fabrication process initially depicted by Douglas et al.15 with changes aimed at reducing overall process duration, while increasing the yield and purification rates.

<table>
	<tbody>
		<tr>
			<td>DNase/RNase free distilled water</td>
			<td>Gibco</td>
			<td>10977</td>
		</tr>
		<tr>
			<td>M13mp18 ssDNA scaffold</td>
			<td>NEB</td>
			<td>N4040S</td>
		</tr>
		<tr>
			<td>10x TAE</td>
			<td>Gibco</td>
			<td>15558-042</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 mL centrifugal filter 100K MWCO</td>
			<td>Amicon</td>
			<td>UFC510024</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Promega</td>
			<td>V3125</td>
		</tr>
		<tr>
			<td>TBE buffer</td>
			<td>Promega</td>
			<td>V4251</td>
		</tr>
		<tr>
			<td>Ethidium bromide 10mg/ml solution&#160;</td>
			<td>Sigma Aldrich</td>
			<td>E1510</td>
		</tr>
		<tr>
			<td>1 kb DNA marker</td>
			<td>NEB</td>
			<td>N3232S</td>
		</tr>
		<tr>
			<td>Loading Dye</td>
			<td>NEB</td>
			<td>B7021S</td>
		</tr>
		<tr>
			<td>uranyl formate</td>
			<td>polysciences</td>
			<td>24762</td>
		</tr>
		<tr>
			<td>carbon-coated TEM grids&#160;</td>
			<td>Science services</td>
			<td>EFCF400-Cu-50</td>
		</tr>
		<tr>
			<td>Thermal Cycler c1000 Touch</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
		<tr>
			<td>Glow Discharge K100X</td>
			<td>Emitech</td>
			<td></td>
		</tr>
		<tr>
			<td>UV table Gel Doc EZ Imager</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM FEI-G12</td>
			<td>Tecnai</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Representative results are shown in Figure 2A. All lanes contain 1 &#181;g of total DNA, measured via spectrophotometer (OD260). Compared with the circular single-strand DNA scaffold (Lane 2), nanorobots are hindered in the gel due to their higher molecular weight, the result of staples hybridization to the scaffold DNA (Lane 3. Red arrow). The low molecular weight band in Lane 3 represents excess staples which did not bind to the scaffold DNA (Green arrow). After purification via centrifugal ...

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

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

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 ...

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14.4K Views.  Bar-Ilan University. 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). 

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

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

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 ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...

Assembly of Gold Nanorods into Chiral Plasmonic Metamolecules Using DNA Origami Templates

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic nanostructures. The high spatial precision of a DNA origami-templated assembly allows controlling the coupling between plasmonic resonances of individual particles and enables tailoring optical properties of the constructed nanostructures. Recently, chiral plasmonic systems attracted a lot of attention due to the strong correlation between the spatial configuration of plasmonic assemblies and their optical responses (e.g., circular dichroism [CD]). In this protocol, we describe the whole workflow for the generation of DNA origami-based chiral assemblies of gold nanorods (AuNRs). The protocol includes a detailed description of the design principles and experimental procedures for the fabrication of DNA origami templates, the synthesis of AuNRs, and the assembly of origami-AuNR structures. In addition, the characterization of structures using transmission electron microscopy (TEM) and CD spectroscopy is included. The described protocol is not limited to chiral configurations and can be adapted for the construction of various plasmonic architectures.

We describe the detailed protocol for the DNA origami-based assembly of gold nanorods into chiral plasmonic metamolecules with strong chiroptical responses. The protocol is not limited to chiral configurations and can be easily adapted for the fabrication of various plasmonic architectures.

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic ...

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 ...

Single-Molecule SERS Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) SERS is able to provide molecule-specific spectroscopic information about individual molecules and therefore yields more detailed chemical information than other SM detection techniques. At the same time, there is the potential to unravel information from SM measurements that remain hidden in Raman measurements of bulk material. This protocol outlines the SM SERS measurements using a DNA origami nanoantenna (DONA) in combination with atomic force microscopy (AFM) and Raman spectroscopy. A DNA origami fork structure and two gold nanoparticles are combined to form the DONAs, with a 1.2-2.0 nm gap between them. This allows an up to 1011-fold SERS signal enhancement, enabling measurements of single molecules. The protocol further demonstrates the placement of a single analyte molecule in a SERS hot spot, the process of AFM imaging, and the subsequent overlaying of Raman imaging to measure an analyte in a single DONA.

Author Spotlight: Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas  

This protocol demonstrates single-molecule surface-enhanced Raman scattering (SERS) measurements using a DNA origami nanoantenna (DONA) combined with colocalized atomic force microscopy (AFM) and Raman measurements.

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) ...