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><tbody><tr><td>Eppendorf epT.I.P.S. Reloads, capacity 2-200&amp;nbsp;&amp;mu;l</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&amp;nbsp;&amp;mu;l</td><td>A. Daigger &amp;amp; Company, Inc.</td><td>EF8960F-3120000054 EACH</td><td>Adjustable Volume</td></tr><tr><td>Research Plus Pippete - Single Channel - 2-20&amp;nbsp;&amp;mu;l</td><td>A. Daigger &amp;amp; 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&quot; x 300&quot;, 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&quot; (50 mm), 60 mm x 11&amp;nbsp;mm</td><td>Electron Microscopy Sciences</td><td>64917-2</td><td>6 per pack</td></tr><tr><td>6&quot; Wafer, P-type, &amp;lt;100&gt; 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 &amp;amp; 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&quot;</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>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>MultiMode Nanoscope IIIa</td><td>Veeco Instruments, Inc.</td><td></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...

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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

Research

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Chemistry

14.3K 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

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

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

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

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

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

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Biology

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Biochemistry

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

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

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

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

Genetics

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Bioengineering

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 ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

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) ...

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

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