(191f) Directing the Self-Assembly of Multiple DNA Nanostructures in a Single Reaction
AIChE Annual Meeting
Monday, October 30, 2017 - 3:15pm to 4:45pm
Currently, DNA origami molecular self-assembly limits researchers to produce each component separately before connecting them to form a higher order nanostructure. This procedure usually results in low yields of the final assembly and is more time consuming. Being able to introduce a standard multi-component one step folding assembly procedure would significantly progress the field by generally improving the efficiency of nanostructures, increasing the yield of complex assemblies, and reducing the overall manufacturing costs in terms of time and materials. In addition, a better understanding of the folding dynamics of DNA origami self-assembly and the establishment of improved assembly control strategies can greatly impact the field of DNA origami and biomolecular nanotechnology.
Structures are usually heated to 65°C and slowly cooled. The critical temperature at which the majority of the self-assembly process occurs is the annealing temperature. Controlling the annealing temperature and rate of DNA nanostructure formation is critical to understanding folding dynamics and manufacturing higher order DNA nanostructure assemblies. The design of staple sequences and length can control DNA binding thermodynamics, which is manifested through the assembly, or DNA annealing, temperature. DNA staple concentrations can control the DNA binding kinetics, which is manifested in the rate of folding. Previous findings have reported the domains/components of a single structure can be controlled by both the annealing temperature of the staples and the concentration ratio of staples within that domain [10-14]. Thus, we will exploit the ability to control thermodynamic and kinetic factors for individual structures in order to optimize the combined folding of multiple structures.
The main research goal is to establish robust methods to fold two distinctly different DNA origami structures in one reaction and to exploit control over the thermodynamics and kinetics of the DNA origami self-assembly process to optimize folding of a combination of structures and enable control over the relative yield of each structure. Building on previous research, the effect of varying staple concentration on the thermodynamics and kinetics of folding will be investigated. Correlations between these fundamental parameters for individual structures will be used to help understand and predict how a mixture of structures will fold. We hypothesize that multiple structures can fold in a single reaction with high overall yield and controllable relative yield by individually controlling the kinetics and thermodynamics of each structure. We tested the possibility of folding two structures with similar but distinguishable geometry, in the same folding reaction where the staples for both structures compete for the same scaffold, and we explored the effect of adjusting the staple concentration of each structure during folding.
In order to investigate the thermodynamic behavior of a DNA origami nanostructure we followed a three-step procedure. First, each structure is heated to 65°C and later annealed at temperatures between 60° and 40°C to determine its annealing temperature. We characterize the structures independently using standard methods such as gel electrophoresis and transmission electron microscopy (TEM). Gel mobility shifts indicate conditions at which structures fold and band intensities provide relative yields. TEM verifies the quality of fold and can be used to characterize undesired structure formation (chimeras). Second, we folded two structures with similar annealing temperature and with equal staple concentrations in order to determine the more thermodynamically favorable structure. Third, we will keep the relative staple concentration of the more stable structure constant and vary the staple concentration of the other structure to determine how this affects the folding efficiency of both structures. By adjusting the staple concentrations we can tip the balance toward formation of either structure and adjust relative yields.
To investigate the folding kinetics of these DNA origami nanostructures we followed a two-step procedure. First, we folded each structure at eight different time points, varying from 5 minutes to 3 days, at the annealing temperature of each individual structure. To ensure that no folding occurs after a desired time point we arrested the folding by flash freezing samples in liquid nitrogen. This way we characterized the individual folding kinetics for each structure separately. Second, we repeated the procedure in step one but with a combination of the two similar structures. We repeated this step for a range of relative concentration combinations to fully characterize the kinetic behavior and the impact of varying the relative concentration ratio. Characterization by gel electrophoresis and TEM was employed to determine how kinetics of individual structures correlate with yields in the combination folds.
Interestingly, we find that chimeras that form over a short timescale appear to revert to well folded structures over long time periods. These findings establish a foundation to assemble complex multi-structure assemblies in one step, and this work advances the field of nanomanufacturing by establishing thermodynamic and kinetic principles for the controllable and scalable self-assembly of an entire fleet of nanostructures simultaneously.
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