The authors thank Dr. Gary Bernstein for use of the AFM.

1. Experiment Planning and Material Preparation
<ol>
	<li>Determine the design, concentration, and functionality of the DNA origami that will be used in the experiments.14-16 Here, we use a DNA origami rectangle design prepared in 1x TAE/Mg2+ solution (40 mM Tris-base, 20 mM acetic acid, 2 mM EDTA and 12 mM magnesium acetate, pH 8.0).17</li>
	<li>Autoclave all tips, tubes, and containers to be used. These materials must all be autoclave compatible.</li>
	<li>Prepare a supply of sterile water for rinsing. Fill a sterile jar with approximately 500 ml of 18 M&#937; x cm water, place on a hotplate, boil for 5 min with the cap off, and take the jar off of the hotplate and let cool with the lid placed on the container but not tightened. Store in the refrigerator and prepare a new supply each month or when necessary.</li>
</ol>
2. Preparing the Mica Substrate
<ol>
	<li>Cut substrates to the appropriate size (1 cm x 1 cm squares) with scissors. Mica is thin and brittle. Alternatively, purchase mica in disc form that does not require cutting.</li>
	<li>Cleave the mica using double-sided tape. Mica is composed of layers of minerals separated by intercalating ions, each layer can be peeled off when adhered to double-sided tape.18
	<ol>
		<li>Place the mica squares on the double-sided tape still in the tape dispenser, making sure it is adhered firmly to the tape. Carefully slide the tweezers between the mica and the tape, the top-most layer will be removed and remain on the tape. Immediately after the removal, the mica square will have its clean side facing downward. Make sure to flip the mica over before storing in a container.</li>
		<li>Repeat this three to four times to ensure complete removal of the top most layer and adequate cleaning.</li>
	</ol>
	</li>
	<li>Alternatively, adhere the mica to a container or table-top with a piece of double sided tape and use a second piece to stick to and peel off the top most mica layer. The mica will be properly cleaned in both cases, although the alternative method makes depositing DNA and rinsing and drying the sample difficult due to the second adhesion to the support.</li>
</ol>
3. Depositing DNA Origami on Mica
<ol>
	<li>Briefly, mix the vial of DNA origami using a vortex mixer to ensure even dispersal of nanostructures in solution.</li>
	<li>Pipette 4 &#181;l of solution onto the mica, ensuring that the pipette tip does not touch the substrate. Leave the DNA origami on the mica for approximately 10 min to ensure adequate coverage. The deposition time will vary depending on the concentration of DNA origami used as well as the desired coverage (Figures 2 and 3).
	<ol>
		<li>Rinse the DNA origami solution off of the mica substrate using 100 &#181;l of sterile water over a sink or other liquid receptacle. Pick up the mica using tweezers. Pipette the water on the substrate, with the flow of the drop towards the tip of the tweezers. Shake the mica with a sharp motion downwards to remove the excess water. Hold the tweezers upright so that the water will flow toward the tweezers to avoid contamination of the sample.</li>
	</ol>
	</li>
	<li>Dry the substrate with a steady stream of nitrogen (N2) for 1 min. Make sure that any excess water is removed. Repeat the rinse with an additional 100 &#181;l of sterile water. Dry the substrate with N2 for an additional 3 min. A completely dry substrate is necessary for successful atomic force microscopy (AFM) analysis (Figure 4).</li>
	<li>Analyze the substrate using AFM or store in a closed container.</li>
</ol>
4. CMOS/Silicon Cleaning Set-up
<ol>
	<li>CAUTION: When using the CMOS set-up, use personal protective equipment at all times. The reagents include strong acids, strong bases, hydrofluoric acid (HF), and strong oxidizing agents which can react with waste solvents if reagents are not properly disposed of. Adhere to the following safety precautions:
	<ol>
		<li>House the CMOS bench in a chemical hood with no other processes or set-ups.</li>
		<li>Wear nitrile gloves, lab coat, safety goggles, large industrial nitrile gloves, a spill apron, and a face shield at all times when using the CMOS bench.</li>
		<li>Use plastic tubs as secondary containment when solutions are prepared.</li>
		<li>Use an inert fluorinated polymer measuring beaker for handling the concentrated HF.</li>
		<li>Make calcium gluconate ointment available as first aid for any skin exposure.</li>
		<li>Only allow properly trained personnel to perform the process.</li>
		<li>Always ensure another member of the lab is present in case of emergency.</li>
		<li>Keep MSDS information for all chemicals near the hood.</li>
		<li>Be familiar with the institution&#39;s or company&#39;s chemical spill and exposure policies. 
		Note: HF readily permeates skin and is a calcium scavenger, affecting bones and damaging nerves if exposure occurs. Dermal exposure to a few milliliters of concentrated hydrofluoric acid can be dangerous and even fatal. Establish necessary precautions to ensure exposure does not occur.</li>
	</ol>
	</li>
	<li>Perform RCA1 and RCA2 in a separate 250 ml glass beakers on separate hotplates. Each beaker should contain a stir bar. Monitor the temperature of the solution using thermometers clamped so the stir bar does not bang into the bulb. Cover the beakers using a watch glass to diminish the effects of evaporation.</li>
	<li>RCA1 Preparation
	<ol>
		<li>Place 50 ml of 18 M&#937; x cm water into the designated RCA1 beaker using a measuring beaker.</li>
		<li>Add 15 ml of concentrated ammonium hydroxide (NH4OH) to the beaker. Rinse the measuring beaker with 25 ml of water and add the rinse water to the RCA1 beaker.</li>
		<li>Turn on the heat and stirrer on the hotplate and bring the RCA1 bath to 70 &#176;C.</li>
		<li>Add 15 ml of 30% hydrogen peroxide (H2O2) to the RCA1 beaker. Use the RCA1 solution within 1 hr after the H2O2 has been added. The bath can be used several times within the span of three days if 15 ml of peroxide is added to the bath each time.</li>
		<li>Rinse the measuring beaker thoroughly with water and discard the rinse in an appropriate RCA1 waste bottle.</li>
	</ol>
	</li>
	<li>RCA2 Preparation
	<ol>
		<li>Add 70 ml of 18 M&#937; x cm water to the designated RCA2 beaker using the thoroughly rinsed measuring beaker.</li>
		<li>Add 15 ml of concentrated hydrochloric acid (HCl). Rinse the measuring beaker with 20 ml of water and add it to the RCA2 beaker.</li>
		<li>Increase the heat and stir speed of the hotplate until the solution reaches 70 &#176;C.</li>
		<li>Add 15 ml of 30% H2O2. Like the RCA1 bath, use this solution within 1 hr from when the H2O2 is added; additionally, the bath can be reused several times within the span of three days if 15 ml of H2O2 is added before each use.</li>
	</ol>
	</li>
	<li>HF Solution Preparation
	<ol>
		<li>Place 50 ml of water in an inert fluorinated polymer beaker.</li>
		<li>Measure 4 ml of concentrated hydrofluoric acid (49%) in the plastic measuring beaker and add it to the inert fluorinated polymer beaker.</li>
		<li>Rinse out the plastic measuring beaker with a total of 50 ml of water, adding the rinse water to the HF beaker. Wash out the measuring beaker thoroughly with water and discard the washings in a designated HF waste container.</li>
	</ol>
	</li>
</ol>
5. Preparing and Cleaning the Silicon Substrate
<ol>
	<li>Cutting silicon wafers into chips
	<ol>
		<li>Identify the perpendicular and parallel lattice directions on the flat polished surface of the silicon wafer. These directions are used to help make cleaving squares easier. The following instructions pertain to cleaving silicon &#60;110&#62; and may not be suitable for other crystal orientations.</li>
		<li>Place the silicon wafer polished-side-up on a soft surface, such as a napkin. Using the diamond-tipped scribe pen, gently nick the bottom of the wafer along the primary flat edge. Place a small wire, such as a paperclip, below the nick and gently apply pressure to the wafer by placing fingers or tweezers on either side of the nick and pushing down. Doing this will separate the wafer into two halves along the crystal lattice line in the natural cleave direction.</li>
		<li>On another napkin, with a pencil and ruler, measure out the desired width of the squares by marking dots on both the top and the bottom of the napkin. Connect these dots with straight lines. This will serve as a guideline for even square shapes.</li>
		<li>Place one of the wafer halves edge-first between the measured lines on the napkin flushed against the line and repeat the in step 5.1.2 The freshly broken perpendicular pieces should now be the width of the cleaved squares. Turn the wafer horizontally on the napkin. Place it between the perpendicular lines and repeat the process in step 5.1.2.</li>
		<li>Store the freshly cleaved wafer chips in a clean vial filled with DI water to prevent scratching. The silicon chips can be stored indefinitely, but should be cleaned before starting experiments.</li>
	</ol>
	</li>
	<li>CMOS Cleaning of Silicon
	<ol>
		<li>When the RCA1 solution has reached the appropriate temperature, submerge eight to ten 1 cm x 1 cm silicon chips in the solution using an inert fluorinated polymer basket with a 2&#34; diameter. Bubbles of oxygen will form on the chips and beaker walls. If no bubbling occurs, the H2O2 is degraded. Leave the chips in the solution for 10 to 20 min, agitating the basket up and down every few minutes to keep the chips from sticking together.</li>
		<li>Lift the basket containing the silicon chips up and drain well. Move the basket over to the waste beaker and rinse thoroughly with 18 M&#937; x cm water. Immerse in the wash beaker and jiggle up and down for 20 sec. Drain the basket and rinse thoroughly with water over the waste beaker. Empty the waste beaker into a designated RCA1 waste bottle and refill with water.</li>
		<li>After RCA1 cleaning is complete, place the basket into the 1:50 HF beaker for 10 to 20 sec. Use a gentle up and down motion to mix the chips and HF. Lift the dunk bucket to allow the HF to drain completely away. 
		Note: The chip surfaces should be hydrophobic; water will not wet the chip, but instead form droplets with high contact angles. This indicates that the silicon oxide has been etched away and the chip is now terminated by Si-H bonds.</li>
		<li>Place the wash beaker and rinse beaker in a plastic tub, move the basket over the rinse beaker and rinse with 18 M&#937; x cm water. Submerge the basket into the wash beaker and agitate for 20 sec.</li>
		<li>Complete a second drain and rinse cycle with 18 M&#937; x cm water. Dump the wash water into the rinse beaker and refill the wash beaker with water. Pour all waste into a designated plastic HF waste bottle.</li>
		<li>When the RCA2 solution has reached the appropriate temperature, submerge the silicon chips in the solutions using the basket. Leave in the solution for 10 to 20 min. The chip will now be hydrophilic due to growth of a thin (1-2 nm) oxide film.</li>
		<li>Remove after the appropriate amount of time and follow the same rinsing procedures as for RCA1. Dispose of the waste in the appropriate RCA2 waste container. Remove each chip from the basket with plastic tweezers, rinse with water, and blow dry with nitrogen.</li>
		<li>Store chips in a plastic wafer box or in a vial of 18 M&#937; x cm water. The silicon will remain clean when stored in water for approximately three days after cleaning. Make sure that the work area is properly cleaned up and the exterior of the CMOS gloves have been washed. Leave the CMOS gloves in the hood to dry.</li>
	</ol>
	</li>
</ol>
6. Depositing DNA Origami on APTES-functionalized Silicon
<ol>
	<li>Self-assembled Monolayer Formation on Silicon
	<ol>
		<li>Warm APTES to RT before opening. If the bottle is too cold, condensation may occur, causing hydrolysis of the APTES during storage. Add 1,980 &#181;l of 18 M&#937; x cm water and 20 &#181;l of APTES to a clean scintillation vial and swirl to mix. Use this solution immediately.</li>
		<li>Place a cleaned silicon chip reflective-side-up in the scintillation vial, cap it, and let sit for 20 min. Remove the chip using tweezers and rinse with 200 &#181;l of water and dry for 1 min with a stream of N2.</li>
	</ol>
	</li>
	<li>Depositing DNA origami on APTES Functionalized Silicon 
	Note: The steps for depositing DNA origami on functionalized silicon are analogous to those for depositing on mica.
	<ol>
		<li>Briefly mix the DNA origami vial and pipette 4 &#181;l of solution onto the silicon substrate. If necessary, increase the volume of DNA origami solution used to cover the entire substrate as the functionalized silicon is more hydrophobic than the mica substrate. Use a glass cover slip to press the deposition solution down and prevent evaporation during long depositions.</li>
		<li>Let the solution stand for the amount of time necessary for the concentration used and the coverage desired (see Figures 2 and 3 for the effect of time and concentration on surface coverage). Rinse the substrate with 100 &#181;l of sterile 18 M&#937; x cm water and dry with N2 for 1 min.</li>
		<li>Repeat the rinsing with an additional 100 &#181;l of sterile water and dry the substrate with N2 for 3 min.</li>
		<li>Store the sample in a clean container until further experiments or imaging can be performed. Samples begin to show particulate accumulation after approximately one to two weeks of storage depending on how much they are handled.</li>
	</ol>
	</li>
</ol>
7. AFM Imaging and Image Analysis of DNA Origami Samples
<ol>
	<li>Use AFM in Tapping mode in air for imaging purposes. Tapping mode ensures that minimal force will be applied to the fragile nanostructures, compared to contact mode. 
	Note: The imaging parameters will be dependent on the instrument. All images presented were captured using a MultiMode Nanoscope IIIa.</li>
	<li>Select AFM probes for non-contact/tapping mode in air, with gold reflective coating, a resonant frequency (nominal) of ~300 kHz, force constant of 40 N/m, and tip radius &#60;10 nm.</li>
	<li>Process and analyze the AFM images using NanoScope Analysis Software. Perform coverage calculations using ImageJ.19</li>
</ol>

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The authors have nothing to disclose.

There are several steps that need to be emphasized to attain consistent and ideal results. For mica samples, following a strict and thorough rinsing and drying regime, as in steps 3.3 and 3.4, will ensure that high quality images of individual DNA origami can be attained using AFM without the various problems outlined in the Representative Results section. Of primary importance for silicon samples is the cleanliness of the substrate. Following the cleaning procedures outlined in step 5.2 thoroughly and meticulously will ...

First introduced in 2006, DNA origami utilizes the self-assembling nature of DNA oligonucleotides to produce designable and highly ordered nanostructures.1 A myriad of structures have been reported, ranging from smiley faces to latched 3-dimensional boxes.2 DNA origami can be functionalized with various biomolecules and nanostructures, giving rise to research applications in nanoelectronics, medicine, and quantum computing.3 However, the analysis and many future applications are not only dependent on structural design, but also on the adhesion of the DNA origami nanostructures to surfaces. The methods described in this manuscript pertain to the preparation of DNA origami samples on two types of substrates: mica and functionalized silicon oxide.
Mica is the substrate of choice for DNA origami studies because it is atomically flat, with a layer height of 0.37 nm &#177; 0.02 nm.4 It is also easily cleaned, making sample preparation and atomic force microscopy (AFM) studies straightforward. Muscovite mica contains a high density of potassium in each cleavage plane, but these ions diffuse away from the mica surface when in water. To mediate the binding of DNA origami to the mica substrate, Mg2+ is used to reverse the negative charge of the mica and electrostatically bind the DNA phosphate backbone to the substrate (Figure 1A).5 Mixtures of annealed DNA in the presence of large excesses of staple strands give high coverage and good images on mica because the adhesion of DNA origami to the Mg2+-terminated surface is much stronger than the adhesion of single-stranded oligonucleotides (staple strands). Other positively charged ions, including Ni2+ and Co2+ can be used to control the adhesion of DNA on mica.6,7 Changing the concentration of monovalent and divalent cations in solution can mediate adhesion and surface diffusion rates of DNA origami.8 However, the protocol for preparing mica substrates and depositing and rinsing the origami is often not explicitly described in published manuscripts.9 Without a clear protocol, reproducible results can be difficult to obtain.
Mica is an insulator, so it is not suitable as a substrate for some applications in nanoelectronics. Silicon passivated with a thin native oxide has desirable electronic properties, including compatibility with prior complimentary metal-oxide semiconductor (CMOS) processing to create input/output structures and topographic features. Silicon wafers stored in air are passivated with either a thick thermal oxide or thin native oxide film that is relatively dirty, with a high particulate count. Silicon oxide has a much lower surface charge density than mica, and the charge density is highly dependent on oxide preparation and history. At magnesium ion concentrations above 150 mM, good coverages (up to 4/&#181;m2) of rectangular DNA origami can be achieved on oxygen plasma treated silicon substrates; however, this concentration and coverage may change depending on the size and design of the nanostructures being used.10 An alternative protocol for tuning the surface charge is to attach a cationic self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) (Figure 1B) to the oxide. The primary amine on APTES can be protonated at pH values below 9, modifying the charge and hydrophobicity of the substrate.11 For a complete monolayer of APTES to be successfully deposited, the silicon must be appropriately cleaned using Radio Corporation of America (RCA) protocols. These protocols include treatments in ammonium hydroxide and hydrogen peroxide solutions (RCA1) to remove organic residues and particle contaminants. A short etch in aqueous hydrofluoric acid solution removes the native oxide layer along with any ionic contaminants that adhere to the oxide. Finally, samples are exposed to a hydrochloric acid and hydrogen peroxide solution (RCA2) to remove metal and ionic contaminants and form a thin, uniform oxide layer.12 Most cleanrooms have designated hoods for CMOS cleaning protocols, with strict rules about what can be used in these areas. A common problem comes in the form of ions such as sodium, which can disrupt the electronic properties of CMOS structures by creating midbandgap traps.13 Ions commonly used in DNA origami preparation and deposition buffers could contaminate the CMOS baths and cause problems for other researchers using the clean room. For this reason, our group uses a &#39;dirty&#39; CMOS cleaning bench arranged specifically for the small samples used for DNA origami research. This process is a good alternative to the traditional cleanroom set-up and may be suitable for laboratories that do not have access to a cleanroom CMOS bench.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Eppendorf epT.I.P.S. Reloads, capacity 2-200 &#956;L&#160;</td>
			<td>VWR International, LLC</td>
			<td>22491733</td>
			<td>10 reload tray of 96 tips</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes, Polypropylene</td>
			<td>VWR International, LLC</td>
			<td>87003-290</td>
			<td>0.65 mL, natural</td>
		</tr>
		<tr>
			<td>Research Plus Pippete - Single Channel - 20-200 &#956;L</td>
			<td>A. Daigger &#38; Company, Inc.</td>
			<td>EF8960F-3120000054 EACH</td>
			<td>Adjustable Volume</td>
		</tr>
		<tr>
			<td>Research Plus Pippete - Single Channel - 2-20 &#956;L</td>
			<td>A. Daigger &#38; Company, Inc.</td>
			<td>EF8960D-3120000038 EACH</td>
			<td>Adjustable Volume</td>
		</tr>
		<tr>
			<td>Scotch 237 Permanent Double-Sided Tape</td>
			<td>Office Depot, Inc.</td>
			<td>602710</td>
			<td>3/4&#34; x 300&#34;, Pack of 2</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Thermo Scientific</td>
			<td>M37610-33Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Wafer container single, 2&#34; (50 mm), 60 mm x 11 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>64917-2</td>
			<td>6 per pack</td>
		</tr>
		<tr>
			<td>6&#34; Wafer, P-type, &#60;100&#62; orientation, w/ primary flat</td>
			<td>Nova Electronic Materials, Ltd.</td>
			<td>GC49266</td>
			<td></td>
		</tr>
		<tr>
			<td>Powder-Free Nitrile Examination Gloves</td>
			<td>VWR International, LLC</td>
			<td>82062-428</td>
			<td>Catalog number is for size large</td>
		</tr>
		<tr>
			<td>High Accuracy Noncontact probes with Au reflective coating</td>
			<td>K-Tek Nanotechnology, Inc.</td>
			<td>HA_NC/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave Pan</td>
			<td>A. Daigger &#38; Company, Inc.</td>
			<td>NAL692-5000 EF25341C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sol-Vex II Aggressive Gloves, Size: 9-9.5; 15 mil, 13 inch - 1 dz</td>
			<td>Spectrum Chemical Mfg. Corp.</td>
			<td>106-15055</td>
			<td>Before use, rinse with water and scrub together until no bubbles form on the gloves.</td>
		</tr>
		<tr>
			<td>Tweezers PTFE 200 mm Square</td>
			<td>Dynalon Corp.</td>
			<td>316504-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Muscovite Mica Sheets V-5 Quality</td>
			<td>Electron Microscopy Sciences</td>
			<td>71850-01</td>
			<td>10 per pack</td>
		</tr>
		<tr>
			<td>Mica Disc, 10 mm</td>
			<td>Ted Pella, Inc</td>
			<td>50</td>
			<td>Mica discs are optional</td>
		</tr>
		<tr>
			<td>Scriber Diamon Pen for Glassware</td>
			<td>VWR International, LLC</td>
			<td>52865-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation Vials, Borosilicate Glass, with Screw Cap - 20 mL</td>
			<td>VWR International, LLC</td>
			<td>66022-060</td>
			<td>Case of 500, with attached polypropylene cap and pulp foil liner</td>
		</tr>
		<tr>
			<td>4 x 5 Inch Top PC-200 Hot Plate, 120 V/60 Hz</td>
			<td>Dot Scientific, Inc.</td>
			<td>6759-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight-Sided Glass Jars, Wide Mouth</td>
			<td>VWR International, LLC</td>
			<td>89043-554</td>
			<td>Case of 254, caps with pulp/vinyl liner attached</td>
		</tr>
		<tr>
			<td>Standar-Grade Glass Beaker, 250 mL Capacity</td>
			<td>VWR International, LLC</td>
			<td>173506</td>
			<td></td>
		</tr>
		<tr>
			<td>Beakers, PTFE</td>
			<td>VWR International, LLC</td>
			<td>89026-022</td>
			<td>For use with HF</td>
		</tr>
		<tr>
			<td>Shallow form watch glass, 3&#34;</td>
			<td>VWR International, LLC</td>
			<td>66112-107</td>
			<td>Case of 12</td>
		</tr>
		<tr>
			<td>Plastic Storage Container</td>
			<td>VWR International, LLC</td>
			<td>470195-354</td>
			<td>For secondary container</td>
		</tr>
		<tr>
			<td>General-Purpose Liquid-In-Glass Thermometers</td>
			<td>VWR International, LLC</td>
			<td>89095-564</td>
			<td></td>
		</tr>
		<tr>
			<td>High precision and ultra fine tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>78310-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate Faceshield</td>
			<td>Fisher Scientific, Inc.</td>
			<td>18-999-4542</td>
			<td></td>
		</tr>
		<tr>
			<td>Neoprene Apron</td>
			<td>Fisher Scientific, Inc.</td>
			<td>19-810-609</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Gluconate, Calgonate</td>
			<td>W.W Grainger, Inc.</td>
			<td>13W861</td>
			<td>Tube, 25 g</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide 30 % CR ACS 500 mL</td>
			<td>Fisher Scientific, Inc.</td>
			<td>H325 500</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>3-Aminopropyltriethoxysilane</td>
			<td>Gelest Inc.</td>
			<td>SIA0610.0-25GM</td>
			<td>Let warm to room temperature before use.</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide, 2.5 L</td>
			<td>Fisher Scientific, Inc.</td>
			<td>A669-212</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific, Inc.</td>
			<td>A144-212</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td>Hydrofluoric acid</td>
			<td>Fisher Scientific, Inc.</td>
			<td>A147-1LB</td>
			<td>HARMFUL, TOXIC</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultiMode Nanoscope IIIa</td>
			<td>Veeco Instruments, Inc.</td>
			<td>n/a</td>
			<td>Any AFM capable of tapping mode is suitable for analysis</td>
		</tr>
		<tr>
			<td>Dunk basket</td>
			<td>Made in lab</td>
			<td>Made in lab</td>
			<td>The dunk basket was made using the bottom of a PTFE bottle with holes drilled in, PTFE handle, and all PTFE screws.</td>
		</tr>
	</tbody>
</table>

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.

Two variables dictate the coverage of DNA origami on the substrate: solution concentration and exposure time. The adsorption characteristics of DNA origami on mica and APTES functionalized silicon oxide have been previously reported.13 The relationship between the concentration of DNA origami in the deposition solution and the final coverages on mica are summarized in Table 1 and Figure 2, showing increasing concentration results in increased coverage. The time-dependence of b...

Watch this Scientific Journal Video about Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation at JoVE.com

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

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 ...

preparation-mica-silicon-substrates-for-dna-origami-analysis

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14.2K Views.  University of Notre Dame. 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.

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

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 Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

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 ...

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Preparation of Macroporous Epitaxial Quartz Films on Silicon by Chemical Solution Deposition

This work describes the detailed protocol for preparing piezoelectric macroporous epitaxial quartz films on silicon(100) substrates. This is a three-step process based on the preparation of a sol in a one-pot synthesis which is followed by the deposition of a gel film on Si(100) substrates by evaporation induced self-assembly using the dip-coating technique and ends with a thermal treatment of the material to induce the gel crystallization and the growth of the quartz film. The formation of a silica gel is based on the reaction of a tetraethyl orthosilicate and water, catalyzed by HCl, in ethanol. However, the solution contains two additional components that are essential for preparing mesoporous epitaxial quartz films from these silica gels dip-coated on Si. Alkaline earth ions, like Sr2+ act as glass melting agents that facilitate the crystallization of silica and in combination with cetyl trimethylammonium bromide (CTAB) amphiphilic template form a phase separation responsible of the macroporosity of the films. The good matching between the quartz and silicon cell parameters is also essential in the stabilization of quartz over other SiO2 polymorphs and is at the origin of the epitaxial growth.

A protocol is presented for the preparation of piezoelectric macroporous epitaxial films of quartz on silicon by solution chemistry using dip-coating and thermal treatments in air.

This work describes the detailed protocol for preparing piezoelectric macroporous epitaxial quartz films on silicon(100) substrates. This is a three-step ...

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

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.