While the overall project is the sorting of cargos on origami, we expected verification of all of the mechanisms would take longer than one summer. Therefore, this summer was spent verifying three aspects of the project:
Domain Level Design
Overall domain level design is illustrated in figure 1. Following abbreviation will be frequently used: walker [W], walker inhibitor [WI], track 1 [TR1], probe for track 1 [PTR1], track 2 [TR2], probe for track 2 [PTR2], cargo 1 [C1], cargo attacher [CA], probe for cargo attacher [PCA], cargo goal inhibitor [CGI], cargo goal 1 [CG1], probe for cargo goal [PCG], walker goal [WG], and probe for walker goal [PWG].
Random walking playground and cargo sorting playground were designed using a rectangular orgiami.
Sequence Level Design
With our overall design in mind, we must design DNA sequences, down to the base level, which undergo the interactions that we desire, without forming secondary structures and binding in unintended ways. We approach this through a combination of pre-generated noninteracting sequences, and trial-and-error design using NUPACK simulation software.
Verification of Mechanisms through Gel Experiments
Before constructing our origami and observing how it behaves, we run a large number of experiments observable through Gel Electrophoresis to verify that many of our mechanisms behave as we expect them to.
Verification of Mechanisms through Fluorescent Spectroscopy
Various DNA strands were tagged with fluorophores and quenchers in order to investigate different mechanisms more directly, both in solution and on origami.
Verification of Mechanisms through Atomic Force Microscopy
Walkers tagged with biotins were planted onto DNA origami, attempts were made to observe random walking on the origami directly under AFM.
Simulation of Expected Results
Before undertaking our experiments, it's desirable to have an idea what our results our going to look like, particularly in the case of random walking, which we intend to investigate rather thoroughly. To do this, we use a stochastic simulation, written in MATLAB.
Derivation of Random Walk Formula
Besides the MATLAB simulation of random walking and cargo sorting, a random walk formula was developed to further investigate and verify the random walking mechanism on DNA origami. The probability of reaching the walker goal is expressed as a function of the number of steps taken.
Verification of Overall Mechanisms in Solution
All of the essential mechanisms in our system were veriﬁed in solution using polyacrylamide gel electrophoresis. These mechanisms include: walker-track binding, triggering the walker, walking from one track to another, picking up cargo, walking while carrying cargo, triggering the cargo goal, dropping oﬀ cargo, and irreversibly walking from tracks to the walker goal. We are fairly confident that all of these mechanisms work as expected in solution, with a few mysteries that do not seem to interfere with the overall behavior of the system (see Gel Verification).
Verification of Random Walking Mechanism on Origami
We used fluorescent spectroscopy (SPEX) to verify the random walking mechanism on the origami. Goals were tagged with fluorophores whereas walkers were tagged with the corresponding quenchers. Fluorescent signals will decrease when walkers reach their goals. Unlike AFM, which studied individual origami, SPEX experiments studied the collective behavior of all the origami in solution. Hence, the SPEX results were analyzed using both Matlab simulation and mathematical formulae.
We are in the process of using atomic force microscopy (AFM) to verify the random walking mechanism on origami. We plan to image individual origami rectangles (using the 1D random walking playground layout) with an inhibited, biotin/streptavidin-tagged walker at its start site, and verify that walkers begin at their intended start site at one end of the random-walking track. We will then trigger the walker, wait for some amount of time, and image the origami again to verify that walkers have left their start site and are tending to stop at their goal at the other end of the track. This research is still in progress.