Experiments

Reagents

Abbreviation	Full Name	Specifications
Tris	Trizma Hydrochloride	Conc. = 1M, pH = 7.5
AA	Acetic Acid	Conc. = 0.5M
EDTA	Ethylenediaminetetraacetic Acid	Conc. = 0.1M
MA	Magnesium Acetate	Conc. = 0.1M
DNA Working Stocks	Various DNA Strands	Conc. = 1 μM

Procedures for Walker Synthesis

This is the procedure to create the DNA walkers. It is the functional nanoparticle and payload carrier in the system. Depending on the requirements, it can be programmed later to host different attachments such as gold, quantum dots, and so on.

They are prepared from a pre-prepared batch of DNA that has been ordered as programmed strands to assemble the walkers after specific thermocycling. Therefore, the following procedure has to be followed very carefully for the self-assembling phenomenon to occur.

Time Since Start of Work (hrs.)	Instruction
00:00	Remove stock solutions of DNA from the Freezer
00:10	Prepare the buffer solution in a PCR Tube: Add 49.5 μL of milli-Q water to the PCR tube Add 12.5 μL (6+6.5uL) of 100mM Magnesium Acetate to the PCR tube Add 4 μL of 1M Tris-HCl to the PCR tube Add 4 μL of 500 mM Acetic Acid to the PCR tube Add 2.5 μL of 100 mM EDTA to the PCR tube
00:30	Add the following amount of solutions to the PCR Tube (in µL): Walker - 1: 3.9 Walker - 2: 5.3 Walker - 3: 4.3 Walker - 4: 3.3 Walker - 5: 3.9 Walker - 6: 3.9 Walker - 7: 2.9
0:40	Close the PCR tube, tap down any droplets that may be on the sides of the container and vortex for one minute.
0:45	Run qCycle.exe Press the Load/Edit button and open the appropriate walker synthesis script Press Start Program Enter 100 μL into the text box when prompted for the solution volume Allow the PCR machine to run. This process will take approximately 4 hours, depending on the equilibration time of the instrument
4:45	Remove the sample from the thermocycler after equilibration, then freeze it.

Results

The walkers in this experiment turned out well for the experiment, as they were able to fulfill the purpose of hosting the biotinylated DNA, and were also able to bind to the LGNPs. However, our AFM images show that there was significant aggregation visible in the sample, likely as a result of discrepancies in volumes added, as well as some discrepancies with leaving samples outside the freezer for a long duration. The expected size of the walkers was around 10 nm, but the observed size of the particles according to the AFM images is around 80 - 100 nm.

Reagents

• Subscript denotes solvent type

Abbreviation	Full Name	Specifications
Tris	Trizma Hydrochloride	Conc. = 1.0 M, pH = 7.5
AA	Acetic Acid	Conc. = 0.5 M
EDTA	Ethylenediaminetetraacetic Acid	Conc. = 100 mM
MA	Magnesium Acetate Solution	Conc. = 100 mM
DNA	Various DNA Strands	See Table
DDIW	Double Deionized Water	R = 14.0 MΩ∙cm-1

Procedure

This procedure is for the synthesis of the DNA Origami Cassettes. The Cassettes act as “cargo-loading stations” for the Walkers; the Cassettes donate their nanoparticle cargoes to the Walkers when within a certain proximity.

• The Q Cycler was used to thermo cycle the Cassettes
• The program qCycle.exe was used to run the Q Cycler
• VWR 200 µL PCR tubes were used for the reaction

The following table lists the DNA strands required and the required volumes.

Strand-ID	Volume (μL)
C2-BKL	10.7
C2-BS	11
C2-DG	6.7
C2-SET-J1	8.3
C2-SET-J2	10.5
C2-PC1	9.4
C2-SHIELD	8.9
C2-LB	12.4
C2-DR	8.9
C2 - PC2	5.7
C2-P2	8
CX-R1	9.5
CX-R2	9.5
CX-BR	8.9
CX-P1	9.2
CX-BL	8.3

Synthesis Procedure

Time Since Start of Work (hrs.)	Instruction
00:00	Take the DNA strands out of the freezer to allow them to thaw
00:05	Add 8.1 μL of DDIW, 25 μL of MA, 8 μL of Tris, 8 μL AA, 5 μL EDTA
00:10	Vortex the PCR Tube for 30 seconds, then add all of the required DNA Strands listed in the above table
00:20	Vortex the PCR Tube for 1 minute, then place in the thermocycler. Close the thermocycler and run the Cassette program, using 150 μL for the volume input
07:20	Take the PCR tube from the Q Cycler, then store in the freezer after equilibrating to room temperature

Results

Our AFM characterization results show that the cassettes are approximately 40 nm by 70 nm, which are the desired dimensions. The DNA tile is 150 nm by 200 nm, so 3 cassettes can effectively be fit onto the tile. The cassettes will be conjugated with their respective cargoes before proceeding with walking the walker across the tile.