HINGE ASSEMBLY

OVERVIEW

 The goal of single hinge assembly was to establish the conditions for consistent yield of single hinges for downstream experiments, such as purification and extended hinge assembly. To do this, we assembled H1 and H2 single hinge units with and without the presence of locking strands used for structure actuation in subsequent experiments. We then assessed for structure assembly using gel electrophoresis.

BACKGROUND INFORMATION

Single hinge units contain three key components: a p8064 scaffold strand, staple strands, and MgCl2. The p8064 scaffold is a type of scaffold used in DNA origami that acts as the backbone of the structure. For our single hinges, this backbone is folded into bundles of DNA helices, creating the top and bottom brick of the hinge, with part of the scaffold running between the two bricks to create the hinges’ pivot point. The scaffold is folded via many complementary-short staple strand interactions as shown in Figure 1 (1). 

Figure 1: DNA origami folding through complementary binding between a scaffold strand and staple strands (1).

The staple strands are added in a five-fold molar excess to the scaffold to promote the complete folding of the scaffold to a single hinge, which has been used previously for similar structures and is a standard approach used in the field (2,3). MgCl2 is added to stabilize DNA by neutralizing its negative charge though electrostatic interactions and chemical bonds (4). Without MgCl2, repulsion of negative charges between DNA strands may disrupt a DNA structure’s folding. The components for single hinge assembly are put through an annealing ramp that helps promote staple-to-scaffold binding before assessing the structure assembly with gel electrophoresis.

Gel electrophoresis is used to separate molecules, in our case DNA, based on size. In gel electrophoresis, DNA migrates through a porous gel by the application of an electric field between two electrodes. DNA is negatively charged, and thus migrates through a gel from the anode (negative electrode) to the cathode (positive electrode), shown in Figure 2 (5). 

Figure 2: A sample agarose gel electrophoresis set up with molecules, visualized as bands, traveling from the loading wells towards the anode (5).

Smaller DNA molecules travel faster through the gel than larger ones, causing a size separation which can be seen under ultraviolet light as bright bands. Structure assembly and locking mechanism assessments were done using agarose and polyacrylamide gel electrophoresis, respectively.  Due to its capabilities of resolving larger structures agarose gel electrophoresis was used for our single hinge units, whereas a polyacrylamide gel was preferred for small DNA molecules, such as those used in our locking mechanism.

METHODS

We first created a version of the H1 and H2 hinges without the incorporation of any padlock strands used in hinge actuation. To do so, single hinge units were formed by combining p8064 scaffold (20 nM), staple strand master mix (100 nM), 10X folding buffer (1mM EDTA, 5mM NaCl, 5mM Tris) and MgCl2 (20 mM). The staple strand master mix was made with each staple in a 1:1 molar ratio with one another. Table 1 shows the concentration and volumes to make one batch of single hinges, which we scaled accordingly to create larger batches. The same procedure was used to make both the H1 and H2 hinge.

Table 1: Components in a batch of single hinges

After forming the single hinge solution, a 37-hour thermal annealing ramp was performed to promote the DNA folding into the final structure. During the 37-hour ramp, single hinges were heated to 80°C, followed by a cooling rate of 4 °C per hour to 60 °C, a second cooling rate of 1°C per hour to 24°C, and a finally cooled at 1 °C per hour to and held at 4 °C. This protocol was adapted from Douglas et al. (6). 

Agarose gel electrophoresis was used as a preliminary check of single hinge formation. To create a 1% agarose gel, 1 g of agarose was added to 100 mL of  1X TAE with 11 mM MgCl2, and dissolved in a microwave. 10 μL of Sybr Safe was added to the dissolved gel and allowed to solidify at room temperature. Samples were created by mixing 5 μL of the assembled hinge solution with 1 μL of 6X loading dye. For the negative control, p8064 scaffold was diluted to 50 nM in TE buffer, from which 2.5 μL of the diluted scaffold was mixed with 0.5 μL of 6X loading dye. Additionally, 3 μL of a 1 Kb plus ladder was loaded into the gel as a reference to judge migration distance of the samples compared to the negative control. The gel was run for 1.5 hours at 90 V and 1X TAE with 11 mM MgCl2 was used as the running buffer. The gel was then imaged using a GelDoc.

To form single hinge units with the padlock strands incorporated, samples were made as shown in Table 2 and subjected to the same gel conditions as above. These samples were used when evaluating the locking mechanism. Please see [Locking Mechanism] for more details.

Table 2: Components in a batch of single hinges with padlock strands.

RESULTS

Gel analysis showed an upward shift in band location toward a larger molecular weight in lanes that contained assembled H1 and H2 single hinge units compared to the scaffold negative control. Scaffold resolved between 1500 and 2000 bp, whereas single hinges resolved between 2000 and 3000 bp. This difference in band location suggested the assembly of H1 and H2 single hinge units. Excess staple strands not incorporated into the assembly of the hinges were observed as smeared blobs at the bottom of the gel below the 75 bp mark. 

The same banding pattern was observed in gels containing hinges assembled with padlock strands. These similarities suggest that the presence of padlock strands during hinge folding does not significantly impact the formation of single hinge units.

Figure 3: H1 and H2 Before purification with and without padlock strand. Figure 3A) Shows H1 and H2 with padlock strand added in different ratios.  Lane A shows H1 and padlock strand added in 1:2 ratio, lane B shows H1 and padlock strand with 1;5 ratio. Lane C shows H2 with padlock strand in 1:2 ratio, lane D shows H2 with padlock strand with 1:5 ratio. Figure 3B shows H1 and H2 without the padlock strand.

DISCUSSION

The 1 Kb plus ladder was only used to determine differences in band migration between samples and controls. This was because the 2D ladder cannot be used to infer dimensions of a 3D structure such as the hinge. The upward shift of bands containing assembled hinges was used to infer that staple strands had bound to the scaffold and likely assembled into their intended H1 or H2 hinge structure. If assembly was successful, these differences to the negative control would be expected. Proper assembly of H1 and H2 was later confirmed using TEM imaging. These results will be published after the judging period in alignment with competition rules.  

Additional information on the optimization of single hinge assembly parameters can be found in the supplementary information.