The treatment for cancer is commonly based on the use of therapeutic toxic molecules for killing or impeding tumour cell growth. However, the delivery of these molecules does not specifically target the tumour cells, often affecting healthy cells, which results in aggressive side effects to the patients. To overcome these side effects it is necessary to develop delivery methods that are capable of releasing the therapeutic molecules inside the desired cells. As such, it is essential to have delivery vectors compatible with biological milieu (able to withstand the biological pressure to its integrity) and have controlled and targeted release of the therapeutic molecules.
One promising vector can be found in DNA nanotechnology. Based on the self-assembly properties of DNA molecules, DNA nanotechnology allows the rational design of nanostructures with predictable geometry and function. One of the advances with higher impact on this field was the DNA origami method. This method produces nanostructures by assembling a long single-stranded DNA molecule, acting as scaffold, with hundreds of synthetic oligonucleotides (usually 20-50 nucleotides in length) programmed to act like staples holding the scaffold in the pre-designed structure.
The main objective of this project was to produce innovative DNA self-assembled nanostructures with the goal of evolving new approaches for drug delivery. This was to be achieved by introducing new elements into the DNA nanostructures improving their versatility and structural integrity. This project aimed to:
• build and characterize DNA nanostructures using long oligonucleotides of biological production;
• add different elements to the nanostructures, such as gold nanoparticles and proteins to the nanostructures to tune the respective intrinsic and structural properties;
• develop new strategies to simplify the assessment of the integrity of nanostructures using fluorophores;
• apply the newly developed nanostructures to assemble a delivery system for doxorubicin.
The progress of the work lead to the discovery of different issues regarding the production of long oligonucleotides in a larger scale. During the execution of the project, several obstacles were raised to the production of the desired oligonucleotides required to produce the DNA nanostructures. To tackle these obstacles, we needed to study the enzymes used in the MOSIC method, the method we used to produce the single-stranded DNA we required for assembling the nanostructures. We studied the activity of BtsCI and BseGI in the specific circumstances of digesting DNA hairpins and discovered a sequence dependence digestion of DNA that is not described in the literature. This work allowed us to improve the yield of production of DNA.
We tested the inclusion of long DNA staple strands in typical DNA nanostructures with little success. We were able to introduce one long oligonucleotide per structure but the inclusion of more long oligonucleotides led to the reduction in size of the structures, indicating an incomplete assembly. Regarding the remainder objectives, recent work by other groups provided solutions that we considered to include in our nanostructures if we had succeeded in producing long oligonucleotide DNA nanostructures.
