ABSTRACT

A. DNA origami: design, fabrication, and analysis overview

The year 2022 marked the 40th anniversary of the original conception of making synthetic nanostructures out of DNA with the idea of building 2- or 3-dimensional lattices out of many similar copies of nucleic acid junctions (25). In 2006, the development of scaffolded DNA origami (6), which we refer to here as just DNA origami, took a major step in enabling more complex nanostructure geometries with a robust and versatile design and fabrication process. DNA origami is based on folding a long scaffold strand, typically ∼7000 to 8000 nucleotides long, into a compact nanostructure through Watson-Crick base-pairing interactions (26–28) with many, often ∼150 to 200, shorter staple strands that are ∼30 to 50 nucleotides long. The staple strands are designed to be piecewise complementary to the scaffold so that binding pinches, or folds (hence the term “origami”), the scaffold into the desired shape (Fig 1A,B). Those shapes typically consist of several double-stranded DNA (dsDNA) helices connected in parallel into bundles with a prescribed geometry where helices are connected to their neighbors at regular intervals by junctions, similar to Holliday junctions (29, 30), where scaffold or staples cross from one helix to the neighboring one (Fig 1C,D). This approach allows for the fabrication of precise nanostructures with dimensions on the 5-to-100-nm scale and unprecedented geometric complexity. Examples include 100-nm-wide smiley faces (6), intricate ∼5- to 100-nm wireframe structures (31, 32), dynamic components like hinges (13, 33), sliders (33, 34), rotors (35, 36), or even ∼150-nm airplanes (21). Here we focused our development of classroom methods for DNA origami fabrication on a previously designed nanorod structure that is in development as a drug delivery device (3). The device is referred to as the “Horse,” as in the original publication, inspired by the concept of the Trojan Horse.

View Figure

• Download as Powerpoint
• Download Figure

The basic DNA origami nanostructure design process (20) follows several common steps: (a) defining the geometry (i.e., cross section in terms of dsDNA helices and the lengths of those helices), (b) scaffold routing, (c) staple routing, and (d) staple sequence determination. These steps are typically carried out using custom computer-aided design (CAD) software. The most widely used software since the development of DNA origami is caDNAno37, and a number of more recent tools have led to faster, partially or fully automated, and more advanced design capabilities (21, 32, 38–40). In addition, significant research over the past decade has led to a number of simulation tools (19, 41–44) that are useful to predict the structure of DNA origami. Figure 1D illustrates a molecular simulation for the small DNA origami rectangular plate example simulated using the oxDNA coarse-grained molecular dynamics model (41, 42). The DNA origami structure of interest for this work, the Horse nanostructure, was designed in the software caDNAno37. Although we circumvent the design process in our educational translation by using a previously published device, this work can still serve as a basis for introducing and learning the DNA origami design process. To facilitate that, we provided a schematic of the Horse structure caDNAno design in Figure S1.

Once the staples are designed, they are typically ordered from one of several commercial vendors who synthesize custom DNA oligonucleotides, and the scaffold can also be purchased from a commercial vendor or produced in a laboratory as previously described (19). Once the scaffold and staples are obtained, the fabrication, or folding, of DNA origami structures is carried out via a temperature-controlled molecular self-assembly process. Commonly used fabrication protocols are described in detail here (19, 23, 45). To fold the structure, the combination of staples strands is mixed in 10-fold excess relative to the scaffold strand, and the mixture is subjected to a thermal folding ramp that consists of 3 phases: a melting phase, an annealing phase, and a cooling phase. Details can vary from structure to structure and are often subject to optimization for individual structures. Generally, the thermal annealing can take many hours or up to several days. For the case of the Horse nanostructure, the original fabrication thermal ramp consisted of melting at 65 °C for 10 min, followed by slow cooling from 60 °C to 25 °C over the course of 17 h, and finally rapid cooling to 4 °C (3).

After fabrication, a common first-step assay to evaluate folding is agarose gel electrophoresis (19). The well-folded compact structures typically migrate through the gel faster than misfolded structures. In most (but not all) cases, the compact folded structure also runs faster than the component scaffold strand, which can be included as a reference. A sharp band that migrates on the gel faster than the scaffold is typically indicative of a well-folded structure. Gel electrophoresis can also serve as a convenient purification approach to separate well-folded from misfolded structures and from excess staple strands. To confirm folding and quantify shape and distributions of conformations, folded structures are subjected to imaging via transmission electron microscopy (TEM) or atomic force microscopy (AFM). Protocols for TEM imaging are provided in the methods and both methods are described in detail here (19, 21, 23, 46).

B. Key barriers to educational translation

This work focuses on eliminating major barriers that make DNA origami fabrication and experiments challenging to perform in educational laboratories and classroom environments. The barriers are related primarily to resources and time required for DNA origami. The first major barrier of resources is due to the equipment needs that range from possibly available (e.g., gel electrophoresis) to unlikely available (e.g., thermocyclers) or impractical (e.g., AFM or TEM) for ready access in educational settings. The barrier of time comes from all stages of the process, including (a) design (designing DNA origami structures can take days to weeks, especially for new designers, and even more recent automated or partially automated tools take time to learn), (b) fabrication (self-assembly reactions can take several hours to prepare and up to days to run the thermal ramps), and (c) analysis (gel electrophoresis typically requires 2 to 3 h to set up and run, and AFM or TEM imaging is likely impractical for most educational settings). In addition to these barriers, the shear complexity of designs and the fabrication process are challenges to educational translation. Here we overcome these barriers to enable the hands-on introduction and use of DNA origami technology in instructional labs and classrooms.

A. Folding DNA origami

The horse nanostructures were folded in a single-pot reaction with 200 nM single-stranded DNA oligos (Integrated DNA Technologies), 20 nM M13mp18-derived scaffolds (prepared in-house as described in Castro et al. (19)), 20 mM MgCl₂ (unless otherwise noted), and a buffer containing 5 mM Tris, 5 mM NaCl (pH 8), and 1 mM EDTA; 1.5-mL Eppendorf tubes were used for classroom folding experiments, and 200-μL Eppendorf tubes were used for laboratory folding experiments. The respective folding equipment and conditions are described below for the laboratory and classroom protocols.

B. Purification of DNA origami

DNA origami Horse nanostructures were purified via agarose gel electrophoresis using the respective gel electrophoresis kit for the laboratory or classroom as described below.

C. Imaging DNA origami

Purified DNA origami Horse nanostructures were suspended in the respective running buffer conditions post-gel electrophoresis at concentrations between 1 and 5 nM. A 4-μL sample droplet was deposited onto a plasma-treated Formvar-coated 400-mesh copper grid (Ted Pella) and incubated for 4 min. The droplet was wicked away on filter paper; afterward, the grid picked up a 10-μL droplet of staining solution containing 2% uranyl formate and 25 mM NaOH and then was immediately wicked away. This was followed by picking up a 20-μL droplet of the same staining solution and incubating for 40 seconds before wicking away on the filter paper. The prepared samples were then dried for at least 20 min before imaging. The structures were imaged at the Ohio State University Campus Microscopy and Imaging Facility on a FEI Tecnai G2 Spirit TEM at 80-kV acceleration.

A. DNA origami design

Designing structures using caDNAno37 can take days to weeks, especially for new designers. Recent automated or semiautomated tools allow for design in minutes (21, 32, 38–40). These approaches still require introducing software, which can take several hours to days to present and learn to operate. We circumvent the design process by relying on the previously published Horse nanostructure (3) (Fig 2A). This also allows students to work with a device that is directly relevant to a key application space for DNA origami, namely, drug delivery. While it is not essential for learning the basics of DNA origami fabrication and analysis, which is the focus here, we envision that the design process could be introduced in parallel as desired.

View Figure

• Download as Powerpoint
• Download Figure

B. Rapid fabrication of DNA origami

The Horse nanostructure fabrication was originally carried out via self-assembly in a thermocycler over the course of ∼17 h. To reduce this time and eliminate the need for costly equipment, we built on recent work demonstrating faster (24, 45) and low-cost (24) methods to fold DNA origami structures. Halley et al. (24) demonstrated that the Horse structure folds well within a range of constant annealing temperatures between 40 °C and 60 °C when annealed for 4 h, and at a particular annealing temperature, the Horse structure can fold in as little as 10 min of annealing. This suggests that folding the Horse structure requires only holding a melting temperature at 65 °C and an annealing temperature at 52 °C, followed by rapid cooling, which could be carried out using equipment such as water baths and ice buckets.

Expanding on this work, we aimed to minimize the folding time without the need for precise temperature control. We tested 2 critical parameters for folding: the annealing temperature and the MgCl₂ concentration. The presence of positive ions is essential to screen the repulsions of the negatively charged phosphate groups on the DNA strands (i.e., charge screening), and temperature regulates the stability of binding interactions between the staples and scaffold, allowing the strands to bind in the most stable configuration. We tested these parameters with a rapid thermal cycle consisting of 5 min at 65 °C, 10 min at a constant annealing temperature (performed in a laboratory thermocycler), followed by rapid cooling to 4 °C. We first tested a range of constant annealing temperatures from 60 °C to 50 °C using a MgCl₂ concentration of 20 mM, which leads to high yield assembly for longer annealing times (24). The folding results were analyzed by TEM (Fig 2B) and agarose gel electrophoresis (AGE), performed with laboratory AGE equipment (Fig 2C). These results show that with this ∼15-min folding protocol, the structures fold at annealing temperatures in the range of 50 °C to 56 °C, with 56 °C showing a decreased yield as indicated by the slightly smeared band. These results suggest that the Horse nanostructure folds in ∼15 min with the highest yields observed in the 50 °C-to-54 °C annealing temperature range.

We also investigated how the ∼15-min folding is affected by varying MgCl₂ concentrations, both to confirm that 20 mM MgCl₂ remains an optimal concentration and as a precursor to the intended laboratory experiment module, which focuses on introducing the concept of screening salt concentrations as a common optimization step for DNA origami fabrication. Horse nanostructures were folded in a laboratory thermocycler at 52 °C with the same ∼15-min thermal cycle, and MgCl₂ concentrations varied from 0 to 35 mM MgCl₂ in 5-mM increments. AGE results (Fig 2D) show that structures begin to form at 10 mM MgCl₂ and fold most efficiently at 15 to 20 mM MgCl₂. At 25 mM and above, structures exhibit increasing aggregation, indicated by the buildup of signal in the wells since large aggregates cannot migrate into the gel.

For both sets of experiments, we confirmed that the leading bands consisted of well-folded Horse nanostructures by TEM imaging. Figure 1B shows a representative TEM image of Horse nanostructures folded at 20 mM MgCl₂ using the ∼15-min folding protocol with annealing at 52 °C, depicting well-folded structures. Combined, these results show that Horse structures can fold with high yield within ∼15 min at 20 mM MgCl₂, including 10 min of annealing at temperatures in the range of 50 °C to 54 °C. While we used 65 °C for melting here, prior work has used up to 95 °C for the melting phase (6, 7), suggesting that precise control of the melting temperature is also not critical.

C. Rapid fabrication of DNA origami with classroom-ready equipment

We aimed to translate this fast and simple folding approach to classroom-ready equipment. Building on Halley et al. (24), who demonstrated DNA origami folding with heated water baths, we developed a folding approach that utilizes 2 hot plates to heat 2 1-L beakers filled with ∼500 mL of water (i.e., water baths) and an ice bucket (or additional beaker) filled with ice and water. The heated water baths were used to hold a melting temperature of ∼65 °C to 70 °C and an annealing temperature of 52 °C to 53 °C, and the ice bucket was used for the final cooling step. Temperatures were monitored using standard laboratory thermometers placed in each water bath. Rather than seeking specific hot plate settings to achieve the correct temperatures, we established a simpler approach that relies on manual control of the temperatures (Fig 3A). Both hot plates were kept on their high (500 °C) setting, and beakers were placed on the hot plate until the water baths reached the desired temperature range. The beakers were then removed from the hot plate when they reached the target temperature range and placed on the lab bench on a piece of cardboard (for some insulation). Beakers were placed back onto the hot plate when they reached the lower end of the target temperature range. With our experimental setup, we determined that placing the beaker back onto the hot plate for 2 to 3 s on the high setting would raise 500 mL of water by about 1 °C. This manual back-and-forth process between the hot plate and the cardboard was used during the duration of the 5-min melting and 10-min annealing steps.

View Figure

• Download as Powerpoint
• Download Figure

Horse nanostructure fabrication was carried out by putting the tube containing the folding reaction solution into the melt water bath for 5 min while it was kept in the target temperature range. Tubes were placed into water baths using foam tube holders thin enough to keep the liquid inside the tube submerged below the water level (Fig 3A). After 5 min, the tubes were moved to the annealing water bath for 10 min while it was held in the desired temperature range. Once 10 min had elapsed, the tubes were transferred to an ice bath for ∼5 min. To reveal better insight into this classroom-ready rapid folding approach, we assessed the folding reaction at several stages during the process—after mixing at room temperature, after the melting phase, and after various annealing times—by assessing a small volume of the folding reaction solution with AGE (Fig 3B) and TEM (Fig 3C). Melt and annealing stage samples were quenched in liquid nitrogen to “flash freeze” the reaction and loaded into gels directly after thawing. TEM images were then taken using gel-purified samples. AGE revealed slower-migrating DNA constructs after mixing, likely due to the scaffold binding staples without forming any well-defined structure, and DNA appeared to be aggregated at the end of the melt phase (stuck in the well), although this may have occurred during sample handling. Both AGE and TEM revealed the basic shape (although not well-defined) folds even with 1 min of annealing, and both 5 and 10 min of annealing led to well-folded structures, which is consistent with prior results (24).

D. Analysis of DNA origami folding using classroom-ready equipment

Traditional laboratory electrophoresis equipment is expensive (∼$1000 to $3000 for a gel rig, including required power supply and light source for visualization), making it impractical for many K-through-12 or undergraduate-level science classrooms. To facilitate broader access, especially for education, some companies have developed inexpensive, safe, and portable gel electrophoresis systems. Here we implemented the MiniOne gel electrophoresis system designed for classroom use (47), which contains a gel rig, power supply, and light source all in 1 compact system at significantly lower cost (<$300 kit; also includes a 20-μL pipette and gel casting equipment). Figure 4A,B compares the equipment needed to set up and run AGE using the MiniOne and research laboratory equipment, respectively.

View Figure

• Download as Powerpoint
• Download Figure

To test the MiniOne electrophoresis system for DNA origami analysis, we performed a salt screen (similar to Fig 2D) using the previously described ∼15-min hot plate water bath folding approach. Since the MiniOne gels contain 6 lanes, we condensed our salt screen to include folding reactions carried out with 0, 10, 20, and 30 mM MgCl₂, leaving room to run a DNA ladder and the ssDNA scaffold as references on the gel. This also conserves materials, which may be an important consideration for educational implementation. The results of these reactions were analyzed by AGE (Fig 4C,D). The MiniOne, however, is designed to run AGE experiments for standard DNA analyses (i.e., not analysis of DNA origami), which are run differently than those typically used for running AGE with DNA origami. DNA origami structures are typically run on 2% agarose gels in a buffer containing 11 mM MgCl₂, both in the gel and in the surrounding running buffer, because positive counterions maintain the stability of DNA origami nanostructures. However, we found that the MiniOne system could not run with these conditions, likely due to the excessive current generated from the high concentration of ions, which tends to heat the gel and running buffer. This heating is normally accounted for by cooling the gels in an ice water bath while they run, but this is not practical in the MiniOne system. We tested a series of AGE conditions to converge to a protocol that was compatible with the MiniOne system, maintained DNA origami stability, and enabled visualization of gel shifts indicative of changes on DNA origami folding quality. The resulting AGE-altered conditions consisted of a 1% agarose gel with 3 mM MgCl₂ in the gel and running buffer. Importantly, the MiniOne gel kits use GelGreen stain for DNA staining, reducing the risks of exposure to the standard mutagenic stain, ethidium bromide, and harmful UV light exposure.

Figures 4C,D compares AGE analysis of the salt screen on the MiniOne setup and laboratory gel equipment, respectively. Both gels illustrate that 0- to 10-mM MgCl₂ leads to misfolded structures. The Horse structure folds well at 20 and 30 mM, but 30 mM also leads to some aggregation, as indicated by the trailing smear in the MiniOne gel and the signal in the well on the laboratory equipment gel.

One additional advantage of the MiniOne system is that the gel migration can be viewed in real time since the visualization is performed directly on the same system as opposed to having to transfer the gel to an imager for the laboratory equipment. Figure 5 shows snapshots of the MiniOne salt screen gel taken at 10-min time intervals (40 min is same as Fig 4C). Structures were visualized using the built-in high-energy LED light source of the MiniOne system, and images were acquired using a smartphone camera. These results illustrate that the relevant gel details (gel shifts indicating well-folded structures and aggregation) can be observed after 30 min. We further confirmed these folding results with TEM imaging of structures purified from the MiniOne gel. Figure 5B shows representative TEM images for each MgCl₂ concentration, confirming misfolded structures at 0 mM, partially folded structures at 10 mM, and well-folded structures at 20 to 30 mM.

View Figure

• Download as Powerpoint
• Download Figure

A. Step 1: preparing the gel for electrophoresis (∼35 min)

Students will prepare the gel running buffer and cast the gel. This involves both standard laboratory measurements as well as pipetting. Preparing the gel takes approximately 15 min. The rate-limiting step here is waiting for the gel to solidify, which takes ∼30 min at room temperature. Students can prepare the folding reaction or bring water baths up to target temperatures while waiting for the gel to solidify.

B. Step 2: running the folding reaction (∼30 min)

In this step, students will prepare the 2 water baths (bringing them to the desired target temperature range), mix the folding reaction, and perform the folding thermal cycle. Students will need to calculate appropriate dilutions to make the different 10× concentrations of a salt buffer (0, 100, 200, and 300 mM MgCl₂) and then mix the 5 ingredients at proper volumes and concentrations (scaffold, staple strands, folding buffer, salt buffer, and water). This process takes ∼15 to 20 min. The rate-limiting step is folding the structure for 20 min (5-min melt, 10-min fold, and 5-min cooling).

C. Step 3: running the gel (∼50 min)

Here, students will perform gel electrophoresis by setting up the gel equipment, mixing the folded structure solution with gel loading dye, loading the samples into wells, and running the gels. Preparation takes ∼10 min, and running the gel takes 30 to 40 min to visualize the gel shift results. Students will compare their results to expected results shown in Figure 4C or D, depending on the electrophoresis setup being used.

The total protocol can be completed in a single, 2-h lab session. If broken up into 2 1-h sessions, the initial session would consist of step 1 and the preparation for step 2 (i.e., mixing folding reactions). The second session would then include running the folding reaction from step 2 as well as running the gel in step 3. Part of the preparation of step 3 (i.e., preparing loading dye) can be done during the folding reaction as long as students can also carefully monitor the water bath temperatures simultaneously. The protocol can also be completed in a single 1-h session if the instructor prepares the gel, water baths, and folding reaction mixtures ahead of time. Students would then perform the folding reaction, mix with loading dye, load, and then run the gel for ∼30 min. This shorter method would be ideal for younger students or students with no prior lab experience.

Table 1 shows the equipment and reagents needed to complete the entire procedure. Many items on the equipment/supplies list (E1 through E12) are commonly found in classroom science laboratories or can be purchased at low cost. E13 through E15 are not as common; however, inexpensive classroom versions exist, such as the MiniOne Gel Electrophoresis Kit used in this research (<$300).

Table 1 Required equipment and reagents.

View Fullscreen

The reagents R1 and R2 are readily found in science laboratories. R3 through R7 can also be purchased at low cost individually or in kits (in these experiments, reagents R3 through R7 were purchased from MiniOne). The reagents R8 through R10 can be provided in small quantities for those interested in performing the procedure.