Design and Fabrication
The design was done iteratively through MagicDNA [9] and caDNAno [10], with simulation as prediction of robustness of the structure in solution. Simulations were done via Structural NUcleic acids Programming Interface (SNUPI) [11] and oxDNA [12]. SNUPI was used for CPU-based rapid screening at the initial design phase for predicting the structure with different scaffold lengths (Figure R1A & R1B), while oxDNA was used for GPU/CUDA-based time step simulations using a coarse-grain model, and different connection profiles were analyzed for mean state (Figure R1C-R1E).
Figure R1: Simulation results from design phase. A. SNUPI plot of TriArm with M13mp18 scaffold. B. SNUPI plot of TriArm with p8064 scaffold. [11] C. oxDNA mean analysis of TriArm-p8064 with crossover connections. D. oxDNA mean analysis of TriArm with straight connections. E. oxDNA mean analysis of TriArm with shortened connections. [12,13]
With finalized design, staple sequences were generated by caDNAno based on a p8064 scaffold sequence, and the corresponding oligonucleotides were ordered from external companies. The initial folding test of the structure was done using a 65-hour folding ramp in a thermal cycler. We tested 2 different versions of TriArm structures with different double-stranded connections in between arms, which are crossover connections (Figure R1C & Figure R2A) and straight connections (Figure R1D & Figure R2B). Structures were tested for folding in different ion conditions, with Mg2+ molarity ranging from 10mM to 24mM. After folding reaction, results were analyzed via agarose gel electrophoresis (AGE) and transmission electron microscopy (TEM) (Figure R2). Results indicate that the structure can be well folded and remain stable with 10-mM Mg2+ present in the solution. A low magnesium concentration is preferred for reducing aggregation as well as maximizing potential for biomedical applications.
Figure R2: AGE (top) and TEM (bottom) results of Mg2+ screening using a 65-hour folding ramp. A. crossover connections. B. straight connections. Scale bar = 100 nm.
Folding of the TriArm structure was further optimized for both ion condition and annealing temperature/temperature range. AGE results in Figure R3 shows the difference of band in terms of band shift and sharpness between properly and non-properly folded structures. We extracted the structures from gel bands for TEM imaging, similar to Figure R2, the structures are partially folded at 6mM magnesium, and the majority of the structures are properly folded at 8mM. A 4-hour isothermal folding ramp was also used for testing the folding temperature/temperature range of the structure (Figure R4). According to the AGE and TEM results in Figure R4, folding begins at below 52.6 degrees Celsius and has a decent yield at 47.7 degrees. In this case, the folding ramp can be set to a shorter time scale from 65 hours to 16 hours, which can be done overnight in order to improve the efficiency of fabrication.
Figure R3: Mg2+ screening with lower range of ion concentration. Scale bar = 200 nm.
Figure R4: Folding temperature investigation using a 4-hour isothermal folding ramp. Scale bar = 200 nm.
Hierarchical Assembly
The TriArm structure was design to be a unit of a reconfigurable higher-order nanomachine assembly. To achieve that, we tested the reconfigurability of the structure in simple assemblies such as linear and polygonal. Figure R5 shows the plausibility of achieving such reconfigurability via changes of connection profile from single-stranded scaffold connection to double-stranded by adding connection strands. The flexibility of single-stranded connections allows a fluctuation of distance and angle between arms, whereas double-stranded connections can be used to enforce a distance of 43 base pairs, ~ 4 turns or ~14.6 nm, assuming 0.34 nm per double-stranded base pair or 3.63 nm per turn in square lattice [8]. By using different combinations of single-stranded and double-stranded connections, the TriArm structure shows different mean angle among its arms based on the oxDNA simulation (Figure R5A & R5B). With this property, we created hierarchical assemblies with oxView and simulated with oxDNA, for a ladder assembly (Figure R5C) and a hexagon formed by 6 complimentary structures (Figure R6).
Figure R5: oxDNA mean analysis results of reconfigurable TriArm. A. arm A and arm B connected double-stranded, other connections single-stranded. B. arms A-B and B-C connected double-stranded, arms A-C connected single-stranded. C. ladder assembly created using structure in A via both A-B and C-C polymerizations. [12,13]
Figure R6: oxDNA trajectory of a hexagon assembly of 6 TriArm structures using A-B polymerization. [12,13]
Due to the end scaffold loops included in the design for prevent stacking, we have designed sequences of sticky ends for connecting scaffold loops of specific arms such as A-B and C-C, which enforce consistency of orientation in contrast with base stacking [14]. Linear assembly of the TriArm version in Figure R5A was performed with A-B connection (Figure R7A), and TEM imaging result indicates an angle change after incubating with connection strands (Figure R7B), which transform structure version shown in Figure R5A into structure version shown in Figure R5B. Figure R5C shows ladder assembly by performing C-C polymerization using linear assembly in Figure R5A. Figure R5D shows a hexagon assembly by turning linear assembly in Figure R5B into fully double-stranded.
Figure R7: Hierarchical assemblies of TriArm. A. linear assembly of structure shown in Figure R5A, with A-B polymerization. B. assembly in A after incubation with connection oligos, which indicates decrease in flexibility during transition from single-stranded to double-stranded (Figure R5B). C. ladder assembly from A, with C-C polymerization. D. hexagon assembly using TriArm structures with all double-stranded connections. Scale bar = 100 nm.
Out-of-plane Investigation
Through the control of connection profile in between arms, we also started the investigation of out-of-plane deformation of the structure using partially double-stranded connections, which created a difference in connection flexibility. This deformation was observed in both oxDNA simulation and TEM imaging. The bend angle is the steepest when the double-stranded connections are at a higher flexibility. As a comparison between Figure R8A and Figure R8B, straight connections have higher flexibility comparing with crossover connections. When all connections are single-stranded, the angle between arms are more randomly distributed (Figure R8C).
Figure R8: Out-of-plane reconfigurability investigation, oxDNA simulation and corresponding TEM images of TriArm structure with A. half straight connections. B. half crossover connections. C. All single-stranded connections. Scale bar = 100 nm.