The first aim of this project was centered around modifying an existing nanostructure. Using caDNAno, poly-T tails were extended off two opposite faces of the box. The new set of sequences were obtained and used to form the structure using a two-day thermal ramp. The success of the formation of the functionalization sites was characterized using gel electrophoresis and TEM imaging. A subtle, yet noticeable shift in the gel was observed due to the increase in molecular weight compared to the original literature box. TEM images confirmed a properly formed structure, however due to a lack of resolution, individual binding sites could not be imaged.
Figure 1. TEM image the successfully folded, purified 2-ended DNA nanostructures.
Our next goal focused on confirming the suggested capping paradigm as well as the actual synthesis of the polymer-cap. A fluorescence experiment was conducted to confirm successful capping. A fluorophore was attached to a poly-A sequence to conjugate with the binding sites. This cap was incubated with the sample for an hour. A gel containing the original literature box with and without the cap, and the 2-ended sample with and without the cap, was run without gel. The gel was then imaged for the Texas-Red fluorophore present in the cap. As expected the only sample to show fluorescence was the 2-eneded structure incubated with the cap. The original box with and without cap and the 2-ended box without cap all showed fluorescent staples present at the bottom of the gel, indicating they had not bound with the sample. The same gel was then soaked in SybrGreen gel dye and imaged for this fluorsescence. The resulting image showed the correct placement of all four samples.
Figure 2. Gel imaged for Texas Red containing (from left to right) 1kb ladder, m13 scaffold, original literature box, the 2-ended box, the original literature box with fluorescent cap, and the 2-ended box with fluorescent cap.
Figure 3. Gel imaged for SybrGreen after being soaked in gel dye. Same samples as Figure 1.
Figure 4. Resultant image from merging fluorescence from Figures 1 and 2.
The peptide cap was then chemically synthesized. The peptide was attached to the complementary oligonucleotide using an inert polymer space to ensure the peptide was not adjacent to the highly charged DNA particle. Proton NMR spectroscopy was used to confirm the successful synthesis. With the capping protocol confirmed, samples were then scaled up and incubated with the new polymer cap to create functional nano-cages.
After successfully forming functional nanostructures, we proceeded to rest the product in vitro. One face of the particle was functionalized with a neuron targeting peptide with a red fluorescent tag on the inner cavity. Separate solutions containing the original non-functionalized box and the new functionalized box respectively, were each administered to their respective primary hippocampal rat neuron samples. After incubation for an hour the samples were fluorescently imaged. In the sample containing functionalized nanoparticles, a significant number of fluorescent cells were observed compared to very few in the control sample. This result proves that not only did our product attach to neuron cell bodies, it attached to the whole membrane making branching extensions visible.
Figure 5. 40X brightfield image of primary hippocampal rat neurons with non-functional DNA box (control).
Figure 6. 40X fluorescent image of primary hippocampal rat neurons with non-functional DNA box (control).
Figure 7. 40X brightfield image of primary hippocampal rat neurons with functionalized DNA box with neuron targeting peptide.
Figure 8. 40X fluorescent image of primary hippocampal rat neurons with functionalized DNA box with neuron targeting peptide.
Prompted by the success of in vitro results, we pushed forward with an in vivo study. The nanostructures were directly injected into the brain of a mouse. The brain slices were compared against a control injection of the peptide-less particles. A significantly larger amount of fluorescence was observed in the slices containing the final functional product. These preliminary results indicated the presence of the functional peptide supported retention of our structures within the brain, while the original particles were circulated out of the brain tissue more rapidly. At this time we were not able to image defined neuron morphologies, however these preliminary results give us optimism that our strategy of targeting the brain from the bloodstream with a dual-targeted nanostructure may be successful.
Figure 9. 10X brightfield image of control sample injected directly into mouse brain.
Figure 10. 10X fluorescent image of control sample injected directly into mouse brain.
Figure 11. 10X brightfield image of functionalized sample injected directly into mouse brain.
Figure 12. 10X fluorescent image of functionalized sample injected directly into mouse brain.