DNA nanotechnology can be used to fold DNA into various shapes with multifunctional purposes. Oligonucleotides can be used to assemble proteins with biosensors and nucleic acid chemistry. Kurt Vesterager Gothelf, a Professor at Aarhus University, explored the significance of functionalizing oligonucleotides during synthesis and the conversion of amine-functionalized oligonucleotides into maleimides.
Azides are chemical compounds characterized by the presence of an azido group. Recent studies have shown that azides can modify the backbone of oligonucleotides. Gothelf and his group wanted to take this research a step further by not only showing how azides can be used for modifying DNA but also for congregating DNA. Azides can react in the middle of DNA synthesis before oxidization occurs. Golthelf explained: “Instead of oxidising, the nucleophilic phosphide, you create this intermediate and then you continue the synthesis deep protect and you end up with an oligonucleotide where you have this modification and you maintain the negative charge as phosphorus.” This modification is highly stable.
Building on this, Gothelf aimed to investigate how different modifications including adding protected alcohols, pyrene dyes, and lipids influence oligonucleotide functionality. Inserting these functional groups creates branched oligonucleotides and even upon adding the functional groups, the melting point of the oligonucleotide does not change much. Gothelf noted that the aim of the modifications is to preserve the negative charge at phosphorous and create stable modifications for oligonucleotide functionalization without compromising DNA duplex stability.
Further studies with Novo Nordisk on using azides for siRNA modifications were conducted to explore delivery efficiencies and knockdown effects. The work revealed that modifications at the prime 5 or ’ ends yielded superior results compared to mid-strand modifications. Comparisons with other conjugation chemistries showed similar knockdown efficiencies, reiterating the robustness of the phosphorus-based approach.
The conversation then shifted to converting amine-modified oligonucleotides directly into maleimides, a process typically cumbersome due to the need for protective groups. By employing specific reaction conditions, the team achieved high-yield conversions, including multi-functionalized oligonucleotides, which were benchmarked against traditional methods for conjugation to proteins.
Gothelf then rounded up the discussion with a few brief insights on DNA nanotechnology, discussing the folding of DNA into complex structures using triplex chemistry. While conventional DNA origami relies on single-stranded DNA, this work innovatively employed double-stranded DNA for enhanced rigidity. Triplex formation via Hoogsteen base pairing was used to assemble various geometric structures, such as helices, squares, and honeycomb patterns. Gothelf remarked: “So we designed a large scaffold with the 9000 base pairs and looked at the folding of this first into a large flat structure and by doing this designing this, we mixed it and it ended up forming structures like this that are pretty large structures that are almost 200 nanometres in width.”