Experimental Structures

For this project, twelve different DNA origami nanostructures were studied. Seven of the structures had 18-double-helix strands while five had six-double-helix strands. Of the 18-helix bundle (hb) structures four of them, the 18hb symmetrical, 18hb seamless, 18hb with nucleators, and the 18hb 42-base pair crossover, were made up of three circular helix rings using the honeycomb lattice (Table 1). The ‘normal’ symmetrical 18hb has internal scaffold crossovers as well as external ones and has up to 21 base pairs between staple crossovers (figure 1-A). The seamless 18hb does not have the scaffold crossover in the middle of the structure only the external ones (figure 1-B),. The 18hb with nucleators has the same scaffold design as the 18hb symmetrical, but the structure has two longer staples(~90 bases long) in the center of the structure to theoretically assist with folding (figure 1-D). The 18hb 42-base pair crossover structure has half of the number of staple crossovers as the 18hb symmetrical leaving up to 42 bases between crossovers, instead of the standard 21 (figure 1-C), increasing the segment binding length and hypothetically trading decrease structural rigidity for increased thermodynamic stability.

Figure M.1: A. 18hb Symmetrical Scaffold B. 18hb Seamless Scaffold C. 18hb 42 base pair crossover staples D. 18hb with Nucleators highlighted in red

The last three 18hb structures all have a different scaffold cross-section design. The flat 18hb is designed with four bundles attached in a line (Table 1). The Douglas (inspired) 18hb has three rows of six double helices, that form four staggered bundles (Table 1). The square 18hb is three rows of 6 helices that is flat and has a square lattice (Table 1). All three structures have a scaffold crossover in the center and have the standard amount of staple crossovers.

Table M.1: Structure Scaffold Lattice Cross Sections

The six helix bundle structures all have a circular double helix ring using the honeycomb structure (figure 2A). The 6hb normal has a scaffold crossover in the middle of the structure and a maximum of 21 base pairs between staple crossovers. The 6hb symmetrical version 2 has the same scaffold structure and staple segment lengths, the only difference is a slightly varied staple pattern (figure 2D). The 6hb with overhangs adds unbound staple segments to the outside of the structure that can be bound to other materials (figure 2E). The 6hb seamless removes the scaffold crossover from the middle of the structure the same as the 18hb seamless (figure 2C). The 6hb with nucleators adds two longer staples to the center to create a nucleation site when folding (figure 2F).

Figure M.2: A. 6hb Solidworks cross section and side view. B. 6hb Normal Scaffold C. 6hb Seamless Scaffold D. 6hb Normal Version 2 E. 6hb with Overhangs F. 6hb with Nucleators highlighted

After performing experiments and matlab analysis of cadnano designs, the group was able to incorporate the features that provided maximum stability into a single structure affectionately named the Rhinocorn (figure 3). Further information on this structure can be found in the Design section.

Figure M.3: A. Solidworks cross section and isometric view of the Rhinocorn.

Methodology

Rapid Fold

With the use of cadnano [1], different sequences of staples were generated to create the various structures tested in this project. The optimal annealing temperature for structure folding was then determined empirically using a rapid folding process. Briefly, scaffold molecules were combined with ssDNA oligonucleotide “staples”, MgCl2, folding buffer (containing Tris, EDTA, and NaCl), and water at a 50 uL scale. The mixture was heated in a thermocycler to 65 ℃ for 15 minutes, held for four hours at various temperatures between 60 ℃ and 40 ℃, and cooled for 15 mins at 4 ℃. The resulting samples were analyzed through agarose gel electrophoresis to determine the temperature that produced the highest degree of proper folding. Each structure’s annealing temperature breakdowns are found in Figure R.2 of the results and discussion section.

Figure M.4: Rapid Fold Gels

Megafold

Once the optimal annealing temperature was determined, the folding process was scaled up to the mL scale. The overall procedure was the same; however, the heating and cooling steps were carried out in heating blocks, water baths, and ice baths. Since this introduced additional variability into the process, the results of the scaled up folding process were validated against a standard fold using agarose gel electrophoresis. This scaled up folding process was used to create a stock of structures on which further thermodynamic, kinetic, and stability experiments could be carried out.

Melt Curve

To gain a better understanding of the thermodynamic properties of our nanostructures, the structures were subjected to high temperatures in order to generate a melting curve. This was achieved using fluorescent SYBR Green intercalating dye and a fluorometer. The structures, at 20 nM concentration, were mixed with SYBR Green at a 1:10 SYBR Green: base pair ratio, and placed in a thermocycler capable of exciting fluorescent molecules. The structures were incubated for 15 mins at 37 ℃ before the temperature was slowly increased (1 ℃ every 1 min) from 37 ℃ to 95 ℃, and the SYBR Green emission intensity was measured. As an intercalating dye, SYBR Green yields a much stronger signal when bound to double stranded DNA versus ssDNA, and so the breakdown of the double helix was recorded as a sharp decrease in fluorescent signal. Melting temperatures and representative melt curves, presented as the negative derivative of relative fluorescent intensity as a function of temperature, can be found in Figure R.2. The melt curves for each structure can be found in the supplementary information.

Folding Kinetics

Kinetic folding experiments were performed to determine how quickly the nanostructures can fold at the optimal annealing temperature. The structures were held at that temperature for 1, 3, 5, 10, 20, 30, or 60 minutes. Once folded for the desired time point, the structures were placed in liquid nitrogen to immediately quench the folding process. Once all the timepoints were carried out, the samples were thawed and immediately run into an agarose gel to determine the minimum time at which the structures were folded. Representative agarose gels and annealing times for each structures can be found in Figure R.2. The kinetics gels for each structure can be found in the supplementary figures.

Figure M.4: Folding Kinetics Gels

Magnesium Stability

Stability of nanostructures in low salt environments was analyzed by performing a magnesium stability test. Briefly, structures were first precipitated from solution by adding an equivolume solution of 15% polyethylene glycol (PEG) (MW=8kDa) and centrifuging at high speeds. The structures were then redissolved in folding buffer containing either 20, 15, 10, 8, 5, 3, 1 or 0 mM MgCl2, and incubated at room temperature for 24 hrs. After incubation, the structures were run in an agarose gel, and the resulting band placements and intensities were analyzed using MATLAB to determine the level of structure breakdown.

Figure M.5: Magnesium Stability Gels

FBS Stability

A similar procedure was carried out to determine structure stability in fetal bovine serum (FBS). After PEG assisted precipitation, the structures were redissolved in a buffer containing either 0, 1, 5, 10, 20, 50, 75, or 100% FBS in clear RPMI cell media (Corning, Manassas VA). After the 24 hr incubation, the samples were precipitated using PEG once again, and resuspended in the standard folding buffer with 20 mM MgCl2 in order to remove the proteins present in PEG before electrophoresis. The band intensities from the resultant gel was once again analyzed using MATLAB to determine the extent of structure breakdown at different FBS concentrations.

Figure M.6: FBS Stability Gels

FBS Spectrophotometry

Spectrophotometer experiments were performed to determine a more specified time frame of the structures break down in FBS and magnesium. The spectrophotometer measures the absorbance of the DNA nanostructures. The baseline for each nanostructure is a 2hour time frame of 100% FBS with measurements taken each minute. The structures that were tested were diluted with 75% FBS to analyze the time frame for the breakdown of the structure. For the magnesium tests, the structures tested were PEG purified, then resuspended in a Tris and EDTA based buffer diluted with magnesium chloride. The amount of magnesium chloride in the resuspension buffer is determined from the salt stability testing.

Reference

• M.1: Douglas, S.D., et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucl. Acids Res. 44(18) (2009)