The primary goal of this experiment was to determine parameters that would lead to the most stable DNA origami nanostructure in physiological conditions that could also intercalate a significant amount of daunorubicin, a commonly used chemotherapy drug. Results of this study could be significant in future design of DNA origami nanostructures for use in in vivo studies and exploring the use of DNA origami as a drug delivery vehicle. Parameters including lattice type (honeycomb or square), surface area, cross section, and daunorubicin retention were assessed to find the optimal DNA origami structure. Prior to daunorubicin intercalation experiments, stability tests were conducted to establish which structures are best suited for a physiological environment in which daunorubicin is used. Stability tests would be able to show which structure would be able to withstand conditions such as enzyme degradation and salt concentrations at the cellular level. Table R.1 below shows a comparison of structure surface area, length, and cross sections. Full analysis of individual structures can be found in the Supplemental section.
Structures were tested under varying magnesium concentrations with a 24 hour incubation period. An equivolume mixture of well-folded structure and 15% polyethylene glycol (PEG-8000) was centrifuged at high speeds to form a pellet. Supernatant was removed and structures were resuspended in buffers ranging from 0 - 20 mM Mg before incubating for 24 hours at room temperature. Gel electrophoresis experiments were performed on the panel of structures with an EDTA-free dye to eliminate chelating effect and make qualitative observations. A MATLAB program was utilized to then further analyze the gels in order to obtain quantitative analysis of structure base pair binding ratio. The MATLAB code was used to measure the intensity of the bands in comparison with the control band to calculate base pair binding ratios.
Table R.2: MgCl2 Stability Results: Concentrations of MgCl2 (mM) at which the following structures begin to degrade and complete degradation: LPP, Branch, Square 18, Horse, and Symmetric 18
The LPP did not show any sign of well-folded structure at 20 mM MgCl2 concentration, with significant aggregation in the well. From 3 mM MgCl2, to 15 mM MgCl2 faint bands can be seen showing slight degradation. Based on the band shift, the gel indicates significant degradation has occurred in the structure between 0-1 mM MgCl2. The branch structure displayed slight degradation at 20 mM MgCl2, while lanes from 3-15 mM MgCl2 indicated structure stability with similar levels of degradation. Gel analysis of the branch also shows significant structure degradation between 0-1 mM MgCl2. Band intensity of the square 18 structure indicated structural stability from 3 to 20 mM MgCl2, with degradation once again occurring between 0-1 mM MgCl2. The horse structure showed little variation in structure stability between 3-15 mM MgCl2, ultimately showing signs of significant degradation between 0-1 mM MgCl2. Lastly, the symmetric 18 (sym 18) structure showed signs of aggregation and degradation at 20 mM MgCl2 , but showed similar levels of stability between 3-15 mM MgCl2 with significant degradation between 0-1 mM MgCl2.
Figure R.2: Normalized Gel Band Intensity MgCl2 Data: Normalized agarose gel electrophoresis intensity graphs for structure panel incubated in 20, 15, 10, 8, 5, 3, 1, and 0 mM MgCl2.
The above gel images were quantified via a MATLAB program that measured the intensities of the folded bands. The following data was normalized relative to the total integrated lane intensity and shown in (Figure R.1). The bar graphs (Figure R.2) show that from a concentration of 3-20 mM MgCl2, the branch and square 18 show that the band intensities remains about the same. The square 18 maintained an average normalized intensity of around 0.30 AU, meanwhile branch sustained a normalized intensity of around 0.45 AU. The symmetric 18, and LPP have a similar trend between the 3-20 mM MgCl2 area in which the structures have a low band intensity at higher salt conditions As the MgCl2 concentration starts decreasing, the normalized average intensity begins to increase reaching its peak. This trend is due to aggregating structures at higher salt concentrations. As the concentration of MgCl2 increases, more structures adhere to each other and cluster together, and this causes the structures to stay in the wells of the agarose gel and not move during electrophoresis. The bands for structures in higher salt concentrations decrease in intensity due to lower amounts of structure moving through the band. A MgCl2 concentration of 3 mM caused the least aggregation in the symmetric 18 and LPP while providing a sustainable environment for the structures to remain folded. This caused the bands for symmetric 18 and LPP structures in 3 mM MgCl2 to have the greatest intensity. All structures tested show a similar trend with the 1 mM MgCl2 and 0 mM MgCl2, where the normalized intensity is close to zero. This suggests that structures only degrade at very low magnesium concentrations.
The panel of structures was tested in differing concentrations of Fetal Bovine Serum (FBS) ranging from 0% - 100%. The presence of FBS tests the limits viable for in vivo settings, in which enzymatic degradation is observed.
Structures were centrifuged after mixing with an equal volume of 15% PEG to pellet the well folded structures. The supernatant was removed and the structures resuspended in the differing concentration solutions of RPMI 1640 and FBS. Afterwards, the structures were incubated for a period of 24 hours in room temperature (25 oC). The structures were once more PEG purified, to remove excess staples. Gel electrophoresis experiments were then conducted to qualitatively analyze the stability of structures.
Figure R.3: 25 ℃ FBS Stability Gels: Gel electrophoresis images of structure panel. From left to right: Ladder, Folded Structure (FS), 0, 1, 5, 10, 20, 50, 75, 100 % FBS at 25oC.
Based on [Figure R.3, i], LPP structure was able to maintain stability up to 75% FBS concentration. Afterwards, signs of degradation were present within 100% FBS as a lower band intensity is observed. The branch structure was able to maintain stability with similar band intensity from 0%-5%, and 75% FBS. However, there was less band intensity between 10%-50%, which can be due to aggregation. Meanwhile, a higher band intensity was observed for 100% FBS for the branch structure. Square 18 showed variable band intensity throughout the various FBS concentrations, with notable signs of aggregation present between 20%-100% FBS wells of the gel. The horse structure maintained the similar band intensity from 0%-100%, with significant amounts of aggregation being present between 1%-100% FBS wells. The symmetric 18 structure was able to maintain stable band intensity until 50% FBS. Signs of degradation were present in 75% and 100% FBS as the bands significantly lose intensity.
To translate the following experiment into quantifiable data, gel images were analyzed via the MATLAB program that was used for the magnesium stability experiment. The program was able to measure band intensity with regards to the corresponding FBS percentages. The square 18 maintained stability from 0%-100% FBS, with normalized gel intensity of around 0.35 AU. Branch was also able to keep a stable gel intensity throughout the FBS concentrations, however there were some deviations within 5-10% FBS illustrating a normalized gel intensity of 0.60, while the other FBS concentrations maintained between 0.45-0.50 AU normalized gel intensity. The low intensity values for the branch corresponding to 0%-1% and 20%-100% FBS can be due to aggregation in those specific wells. The symmetric 18 showed decreasing stability as the FBS concentrations progressed from 0%-100% FBS. LPP kept a low, but steady gel intensity of around 0.25 AU throughout the different FBS concentrations.
The panel of structures was tested in differing concentrations of FBS ranging from 0% - 100%. A volume of structure was centrifuged with an equal volume of 15% PEG to precipitate out the structures. The supernatant was removed and the structures resuspended in the differing concentration solutions of FBS in RPMI 1640. All structures were then incubated for 24 hours at 37℃, to mimic an in vivo environment. They were then centrifuged down again with an equal volume of 15% PEG and resuspended in 50µL of 20mM MgCl2. The stability of each structure was then tested and observed using gel electrophoresis with the corresponding results shown in Figure R.5. Gel images were analyzed with the MATLAB program used for previous and FBS experiments to quantify the band intensities and base pair binding ratios of the structures. The resulting graphs from this analysis are shown in Figure R.5 below:
By analyzing the gel images located in Figure R.5, it can be determined at which percentage FBS each structure is stable. LPP was stable in a solution of up to 10% FBS. As the concentration increased, degradation started to become apparent at 20% FBS with the band completely disappearing at 100% FBS. The branch begins degradation at 5% FBS and becomes more degraded until there is little to no folded structure found in the 75% FBS and 100% FBS lanes. The horse has a consistent band throughout all percentages, but has a band shift downward past 20% FBS indicating a slight degradation of the structure. The square 18 was similar to the horse in that it had a consistent band throughout all percentages of FBS. Square 18 also showed a slight band shift downward beginning at 10% FBS. The sym 18 showed little to no concentration in 0 and 1% FBS which may be due to the aggregation of the structure near the wells. At 10% FBS the whole lane is smeared, indicating large amounts of degradation. At 20% the structure is well formed and from 50% on the bands shift down. All of the structures contain the band shift that trails downward as the percentage of FBS increases. (Degree of degradation based off of intensity is what the graphs show)
Figure R.6: 37℃ FBS Normalized Gel Band Intensity Data: Normalized agarose gel electrophoresis intensity graphs for structure panel incubated in 0, 1, 5, 10, 20, 50, 75, 100 % FBS at 37℃.
The LPP structure showed a downward trend in band intensity starting from 0% FBS to 100% FBS. Despite this, at 100% FBS, some structure was still present. According to [Figure R.6, ii], the branch structure remained stable up to about 75% FBS concentration, with normalized gel intensity being the greatest at 20% FBS, with an average of around 0.58 AU. [Figure R.6, iii] shows that the square 18 maintains a normalized gel intensity of between 0.24-0.42 AU from 0-100% FBS concentration. The symmetric 18 shows a slight upward trend in normalized gel intensity as the FBS concentration increases. The lowest normalized gel intensity for symmetric 18 was around 0.10 AU and occurred at 0% FBS, while the highest intensity of 0.38 AU was present in 50% FBS.
Figure R.7: Structure Concentration vs. BBR Data: Base-pair binding ratios (BBRs) for structure concentrations of 10, 15, and 20 (nM) for the structure panel.
Table R.3: Structure Average BBR Comparison: Outline of average BBRs for the structure panel at 10, 15, and 20 nM structure concentrations.
Each structure was tested to determine its ability in retaining the chemotherapy drug, daunorubicin. The goal of this experiment was to determine the structure concencentration that optimized daunorubicin binding to DNA; binding between DNA origami structures and daunorubicin was quantified by calculating base-pair binding ratios. Binding to more daunorubicin would lead to more efficient drug transport for chemotherapy treatments. Origami structures at 10, 15, and 20 nM were incubated with 250μM daunorubicin. The structures were incubated for 24 hours at a temperature of 37℃. Afterwards, the structures were centrifuged for 15 minutes in order to accumulate the structure as a pellet. The concentration of the daunorubicin in the supernatant was measured to determine the amount of drug that did not intercalate into the structure. The base-pair binding ratio (BBR) of daunorubicin to the structure was calculated through the supernatant concentrations.
The motivation of this experiment was to determine which DNA origami structure will have a higher retention of daunorubicin in a period of 24 hours, and at which concentration of structure would the optimal amount of retention be achieved at. Characterizing the binding ratio will help determine which structure has the greatest potential to be a potential drug delivery vehicle.
The LPP structure yielded a BBR of around 0.60 AU for 10 nM and 20 nM structure concentrations. Meanwhile, 15 nM structure resulted in a BBR of 1.018 AU. The branch structure showed a decreasing trend of daunorubicin retention as the structure concentrations increased. A high BBR of 0.716 AU was achieved for 10 nM structure of the branch. Meanwhile, the branch had a low BBR of 0.487 AU for 20 nM concentration of structure. The square 18 structure showed an upward trend of daunorubicin as the structure concentration increased. Square 18 had the highest BBR of 0.509 AU at 15 nM concentration, while the lowest BBR of 0.299 AU was present in 10 nM structure concentration. The horse structure had its highest BBR of 0.560 AU at a structure concentration of 10 nM. A lower concentration of 0.364 AU is present at 15 nM structure. For the Symmetric 18 structure, 15 nM structure concentration carried the lowest BBR of 0.139 AU. The highest BBR of 0.409 AU was present in 20 nM concentration of structure.