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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - VECTORIGAMI (DNA Origami for drug delivery)

Teaser

Summary

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:
â€¢ build and characterize DNA nanostructures using long oligonucleotides of biological production;
â€¢ add different elements to the nanostructures, such as gold nanoparticles and proteins to the nanostructures to tune the respective intrinsic and structural properties;
â€¢ develop new strategies to simplify the assessment of the integrity of nanostructures using fluorophores;
â€¢ 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.

Work performed

The execution of the project started with the design of DNA nanostructures using the DNA origami method. For the chosen structure, we redesigned the self-assembly to include long oligonucleotides instead of the traditional short oligonucleotides used in DNA origami.
We used the MOSIC method, developed earlier in the group, to produce long oligonucleotides. We came across some difficulties in scaling up the method to produce enough quantity of the high quality oligonucleotides required to include in the DNA nanostructures. To tackle this situation we needed to study the restriction enzymes used in the MOSIC method, BtsCI and BseGI. The results from our study showed the presence of a sequence preference that is not described in the literature. We are now preparing the manuscript related to this discovery that we will submit to an open access journal of the speciality.
After solving the problems with the production of the oligonucleotides we proceeded to the assembly of the DNA nanostructures. We successfully incorporated one long oligonucleotide per structure, with the structures maintaining the same properties as the control short stapled-version. However, the introduction of more long oligonucleotides resulted in the shortening of the structures proportionally, indicating that only exactly one long oligonucleotide per structure was possible. We tried different conditions for the assembly, such as different salt concentrations and different temperature programs, but the success rate did not increase. We decided to try a different method to assemble DNA nanostructures that was developed in our lab and that produced less compact and more spaced nanostructures but the results were similar.
The failure in assembling high quality long DNA nanostructures resulted in a stagnation of the project that delayed the beginning of the remaining work packages.
Regarding the adding of elements to the nanostructures we did some discovery work on adding antibodies to the structures and studied how the spacing of the antibodies in the structures could affect binding efficiency, to improve targeting. The work progressed to study the properties of antibodies and how they interact with ligands at different distances. This work is still undergoing but we foresee the submission of a manuscript for publication by the end of 2017.
Other elements were not tested because the field progressed and other authors published results that were comparable with what was suggestion in this project.

Final results

As was mentioned previously, the progress beyond the state of the art did not result from the main project but from the hurdles we required to overcome to execute the proposed project. We succeeded in including one oligonucleotide into the DNA nanostructures and, as far as we know, we were the first to succeed in adding a 420 nucleotides DNA strand into a DNA nanostructure. Nonetheless, the work developed in the scope of this project did not allow the inclusion of more oligonucleotides in the structures, as was described in one of the objectives of this project. Our results suggest that an improvement in the production methods of long oligonucleotides will be crucial to allow the optimization required to include more than one long oligonucleotide in the structures.
We did progress the state of the art in other areas. We studied the activity of BtsCI and BseGI restriction enzymes and discovered that these enzymes digest preferentially specific sequences, against what is described in the literature. We studied this in both hairpin structures and double-stranded DNA, and we show how these enzymes work with detail.
We also used the DNA origami properties to study the way antibodies interact with antigens at specific distances. Our results expand the state of the art on the comprehension of the flexibility of specific subtypes of antibodies and on how that flexibility relates to their binding efficiency to antigens at different distances.