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Stability Experiments


Based on the results, it is apparent that the most stable of the examined structures are the Horse and Square 18 structures. Many of the structures showed high stability in fetal bovine serum (FBS) (room temperature) and magnesium, with Square 18 showing the highest stability of all structures in magnesium. However the differences in stability are much more apparent when looking at the FBS (37) results. The Horse and Square 18 are the only structures to show slight to no degradation in the gels and band intensity profiles while all other structures began showing considerable degradation starting at 1-5% FBS. An explanation for why these structures have higher stability than the others could be due to their lattice structure. Both Horse and Square 18 have a square lattice while LPP and Sym 18 have honeycomb lattices. Like the other structures, Sym 18 broke down between 0-3 mM magnesium concentrations, but it only showed better stability than the LPP in FBS experiments. Although the Branch did not appear to be as stable as Horse or Square 18, the band intensities were considerably higher than those of the honeycomb lattice structures throughout all of the experiments, indicating a general higher stability. The length of structures did not appear have an effect on structure stability, but increased surface area and the inclusion of overhangs did appear to negatively affect structure stability in FBS. The Horse and Square 18 structures are similarly stable, however they have largely varying lengths and surface areas. Additionally, LPP and Horse have almost identical surface areas while also being hugely variable in their stabilities. From these results, it seems to be that lattice structure has a more significant impact on stability than the length or surface area of structures. In addition, the structures in FBS at 25oC and 37 showed a trend where the bands migrated faster as percent FBS concentration increased. This could be due to small amounts of degradation occurring at the ends of the structures from the nucleases in the FBS. This slight degradation is just enough to preserve the band while also causing the observable shift. Preliminary results of stability experiments suggest that Square 18 and Horse were the most stable structures, indicating that structures with a square lattice that lack overhangs may be more stable in physiological conditions.


Daunorubicin Binding


The ability of structures at varying concentration to bind with 250 𝜇M daunorubicin was largely variable. For each structure, tests were performed with structure concentrations of 10, 15, and 20 nM which corresponds to a daunorubicin to structure ratio of 25,000, 16,667, and 12,500, respectively. Horse and Branch yielded the highest base-pair binding ratio (BBR) at 10 nM, LPP and Square 18 at 15 nM, and Sym 18 at 20 nM. Branch and Horse are very similar structures which may explain why they have the highest base pair binding occur at a concentration of 10 nM. However, LPP and Square 18 are dissimilar in every aspect except both have peak binding at 15 nM. Based on our results it is hard to conclude why different structures have peak binding ratios at varying structure concentrations. Altogether, LPP and Branch had the highest base pair binding ratios of the five structures with LPP having the overall highest binding ratio. Branch base pair binding was higher at a concentration of 10 nM while LPP had the higher binding ratio at concentrations of 15 nM and 20 nM. There is a sudden decrease in binding ratio at 20 nM concentration which could be likely due to saturation of the binding sites while increasing the amount of free daunorubicin. One key distinction between Branch and LPP from the other three structures is that both of these structures have overhangs attached. These overhangs could be one possible explanation for why there is a higher base pair binding ratio at the 10 and 15 nM concentrations. These overhangs may provide additional sites of attachment for daunorubicin in addition to binding to the structure itself, which would explain why Branch and LPP have higher base pair binding ratios than the structures without overhangs. These results indicate that structures with overhangs such as LPP or Branch are more likely to bind increased amounts of daunorubicin compared to structures lacking overhangs. Surface area is also another potential characteristic that may correlate to BBR. The Branch and LPP both have similar surface areas of around 9000-10000 nm2 and had the best BBR. Structures with similar surface areas to Branch and LPP as well as those with very high or low surface areas did not have as high of a BBR. Lattice is also a varying characteristic between structures, however it does not seem to impact BBR. Branch and LPP have the highest BBR and have a square and honeycomb lattice, respectively, while other square and honeycomb lattice structures do not indicate any consistent trends on BBR. This data suggests that the addition of overhangs may have a more significant impact on BBR than other characteristics of the structure such as surface area or lattice.


Structure Choice


Based on the results and conclusions, the Square 18 and the Horse structures were the best suited for daunorubicin retention and stability in physiological conditions. At 10 nM structure concentration, the Horse structure had the highest BBR of 0.56, while the Square 18 had higher BBR than the Horse at 15 nM and 20 nM structure concentration when incubated with 250 𝜇M daunorubicin. As well as daunorubicin binding, the Horse and Square 18 both performed exceptionally well in all of the other tests, however the Horse yielded slightly better results during the 37 FBS tests which more closely resembles an in vivo environment. In these 37 FBS tests, the Square 18 showed an increased amount of structure breakdown between 75-100% FBS when compared to the Horse. Subsequently, the Horse has been chosen as the most stable and suitable out of the panel of structures.