Biomod/2015/OhioMOD/results

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Results
Salt Screen

FIGURE 1. A MgCl2 salt screen. 6 mM was the ideal salt condition found in this 2.5 day ramp. B TEM image of a monomer at 6 mM MgCl2. C NaCl salt screen. 0.6 M was the ideal salt condition found in this 2.5 day ramp. D TEM image of a monomer at 6 mM NaCl. Scale bar 50 nm.


In order to optimize folding conditions for DNA origami, we expect that optimal salt conditions land somewhere between 12-20mM MgCl2 or 0.5-1M NaCl. After designing our structure and ordering our staples, we created our prestocks and working stocks. We then perform a salt gradient experiment in which we put our folding reactions into an 8-strip tube, each with a varying amount of MgCl2, or NaCl. FIGURE (1A) and (1C) shows the resulting purified folding reaction. From here, we chose the optimal salt conditions. As shown in FIGURE (1A) we ultimately chose to go with 18mM MgCl2 for future folding because the bands were cleaner and retained the most structure post purification.

Temperature
Figure 1. A gel image of a DNA origami structure folded from a temperature range of 60° C to 40° C. This gel was run using magnesium at a salt concentration of 18mM.

Figure 2. A thermal ramp over a normal time of 2.5 to 6 days. The temperature of the thermal ranges from 65° C to 23° C. Thermal ramps decreases in temperature by increments of .5° C for every 1-5 hours.


After running a folding reaction to determine the proper salt conditions for our structure, we used that salt condition, 18mM MgCl2, to then determine the best annealing temperature in a rapid fold. While thermal ramps, seen in Figure 2, take anywhere from 2.5 to 6 days to fold DNA origami, rapid folds can reduce that time to as little as 1 to 4 hours total. A thermal ramp heats a folding reaction to 65oC and then steps down the temperature gradient over time. The folding reaction is thus heated for a set period of time starting from 65oC and decreases until it reaches room temperature. A rapid fold, in Figure 3, however, heats the folding reaction to 65oC and then splits the temperature gradient across the 8-strip tube (in this case 60-40oC). Each tube is heated at distinct temperatures for 4 hours and then cooled at 4oC to complete the rapid fold.

Figure 3. A rapid fold thermal ramp in which individual samples in an 8-strip tube are run at different temperatures, to determine the best range of annealing temperatures for a DNA structure. A rapid fold normally takes around 1 to 4 hours.

Figure 4. A normalized intensity plot displaying the results of the thermal ramp used to determine the best range of annealing temperatures for the DNA structure.



To identify the best range of temperatures for folding the structure, a thermal ramp over a temperature range of 60° C to 40° C was used. After completing a thermal ramp it was determined that the optimal folding temperature for our structure was between 52° C and 56° C. This temperature range, seen in Figure 4, was chosen based on the intensity of the bands within 52° C and 56° C.

Figure 5. A rapid fold using the previously determined best range of temperatures for a DNA structure. In this rapid fold the individual tubes are run through a 4° C temperature gradient.


Based on the results of the 60° C to 40° C thermal ramp, a more specified thermal ramp, seen in Figure 5, was run to determine the most optimal folding temperature. A thermal ramp between 52° C and 56° C, with a 4° C temperature gradient, was used to determine the most optimal temperature for folding the structure.

Figure 6. The gel image of the thermal ramp between 52° C and 56° C with a 4° C temperature gradient.

Figure 7. A normalized intensity plot displaying the results of the narrowed temperature range used to determine the best annealing temperature for the DNA structure.


Once the ramp was completed it was determined that 53.8° C is the optimal folding temperature for the structure. This temperature was determined based on the intensity of the bands from the 4° C rapid fold, seen in Figure 6.

Sobczak, J.-P. J., T. G. Martin, T. Gerling, and H. Dietz. "Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature." Science338.6113 (2012): 1458-461. Web. 15 July 2015.

Closing

Hypothesis: The BUS will close in the presence of a single stranded closing DNA that bridge latching overhangs between the top and bottom beams. Addition of an opening strand with greater complementarity to the closing strand will trigger the opening of the BUS.


The BUS must start in a closed configuration so that trimer formation only occurs after detection of a protein (or other biomolecule). Initially, the hinges were subjected to only closing strands, which bind to the latching strands at the end of the hinge. Adding the closing strands resulted in less than 5% closing efficiency.


The zippering strands were utilized, along with the closing strands, to aid closing of the hinges as seen in Figure 6. These strands are 16 bases long; half of the strand is complementary to an overhang sequence of one of the arms while the second half is complementary to an overhang sequence of the other arm (Figure 6A). The closing is dependent on the concentration of zippering strands and closing strands, thus we tested 10x, 100x, and 1000x concentrations relative to the number of binding overhang sites. Figure 6B highlights that closing occurs more efficiently at 10x relative to the number of binding sites; it decreases at 100x and 1000x, likely due to oversaturation of corresponding binding sites. Additionally, closing was tested at both room temperature and 37oC. We found that temperature does not play a significant role in closing.

Figure 6. Hinge Closing (A) Open hinges were subjected to zippering strands and closing strands. The zippering strands binds to the end of overhangs (inset) and closing strands bind to the aptamer strand at the end of hinge. (B) Distribution of percent closed hinges at various concentrations of zippering strands and closing strands. Adding 10x zippering strands results in the most closed hinges (C) TEM images of a closed hinge. Scale bars 50nm.


The closed hinges were PEG purified at 16,000xg for 30 minutes in order to remove zippering strands from structures, as depicted in Figure 7A [1]. For the closed hinges to be functional, they must be able to open in the presence of complementary opening strands. Opening strands were added to a solution of closed hinges at10x, 100x, and 1000x concentrations relative to the number of binding sites. Figure 7C shows that the structures open significantly at 100x and 1000x opening strands relative to the number of binding sites. These preliminary results show that 100x is the most optimal condition for opening; however, we still expect 1000x to be more efficient because opening mechanism allows the oversaturation of opening strand to have great complementarity to the aptamer strands and further tests will need to be conducted to verify these findings. Higher concentrations will promote strand displacement and eventually open up the closed hinges.


Figure 7. Hinge Opening (A) Hinge were closed at 1x zippering and closing strands. The zippering strands were removed using PEG in centrifugation for 16,000xg in 30 minutes. The closed hinges were subjected to opening strands, which displaced and released the closing strands into solution. (B) Distribution of opening hinges at various concentrations of opening strands. (C) TEM image of closed hinges at before the addition of opening strands. (D) TEM image of open hinges at 100x opening strand.


Citations Stahl, Evi et al. “Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions.” Angew. Chem. 126.47 (2014): 12949–12954. CrossRef. Web.

Monomers
After optimizing buffer and rapid fold conditions, we have shown that we have obtained well-folded BUS monomers in sizable quantities. In order to find optimal salt, temperature, and incubation conditions, we varied a single variable at a time while keeping the other two constant. We concluded that our structure is best folded at 18mM MgCl2 and in a 4-hour rapid fold at 53oC.

FIGURE 1. (A) gel image (from left to right: ladder, scaffold, structure in remaining wells) of AB open folded at 53oC and 18mM MgCl2 for 4 hours. (B) TEM image of the structure at 18mM MgCl2 and 53oC. Scale bar 50 nm.


We purified our well-folded monomer structure via gel electrophoresis. We purified three different structures – AB, CA’, and B’C’ – that is, each monomer has a different combination of sets of internal overhangs within the structure. The gel in FIGURE (1) show double bands of structure. Even though we see double bands, we purified and reran each band to understand the difference between each band. Extensive testing (*see supplemental information) concluded that there was no functionally significant difference between each band.


Furthermore, each structure was double PEG purified after rapid folds to get rid of any excess staples. This allowed for a more effective trimerization of monomers in later experiments.

Trimerization
Three factors were important for efficient trimerization – stoichiometric ratio of each monomer, incubation time, and incubation temperature. The best results for trimerization occurred when each monomer was combined in a 1:1:1 ratio and incubated at 37oC for 30 minutes. Below in TABLE (1) there is data considering the percentage of monomers that trimerized at two different incubation times.
Trimer Formation Testing Conditions
TABLE 1. The value of unformed trimers is the number of unattached/un-polymerized monomers divided by three.
At 37⁰C for an incubation time of 30 minutes trimers were able to fold 4x times more than incubating the combined monomers for 4 hours. Long periods of heating appear to be detrimental to the forming or maintaining of trimers. Aggregation of structures occur at long periods of heating.
FIGURE 2. (A) TEM image of a trimer incubated for 4 hours at 37⁰C. (B) TEM image of a trimer incubated for 30 minutes at 37⁰C. Scale bar is set to 50 nm.
Future Work


Short Term Goals


The current condition of the closed and opened BUS structures are not efficient enough for purposeful future work. Therefore, short term goals are implemented and divided into two different sections: optimization of trimer formation and optimization of closing efficiency. The first step in optimizing closing efficiency would include varying incubation time, temperature, and zippering schemes. Fixing the structure in a distinct orientation by increasing the internal overhangs increases the closing efficiency. The second step would be optimizing trimerization by conducting a temperature gradient.


Increasing overhangs for the zippering strands can help to increase the closing efficiency. The second step in optimizing trimerization is (1) testing the temperature in 1o C increments in order to find the ideal annealing temperature and (2) increasing the total number of internal overhangs and increasing their length.


Long Term Goals


Currently, our structure is configured for the proof of concept; that is, we wanted to demonstrate that we can form a higher order structure. The long term goal would be replacing latching strands with aptamer strands and then test whether the BUS undergoes opening under the exposure of VEGF. We also aim to functionalize the BUS interior surface with therapeutic molecules to sequester VEGF, thus suppressing angiogenesis. In the future, the aptamer strand could be implemented to a more specific protein aggregate neurological diseases.









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