| 研究課題/領域番号 |
18K14270
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| 研究機関 | 東京大学 |
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研究代表者 |
P.K. Hashim 東京大学, 大学院工学系研究科(工学部), 特任研究員 (40817277)
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| 研究期間 (年度) |
2018-04-01 – 2020-03-31
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| キーワード | DNA nanostructure / Molecular glue / click reaction / functionalization of DNA |
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| 研究実績の概要 |
For the stabilization and functionalization of DNA-nanostructures using ‘molecular glue’, a novel macromonomer consisting Gu+ ions, benzophenone (BP) and an anchoring azide unit is synthesized and fully characterized. As evident from the gel electrophoresis, the macromonomer adheres to a 200 base pair DNA via BP intercalation and non-covalent ‘salt-bridges’ formations, which is then covalently fixed by a photo-irradiation. The universality of the approach is also tested by using DNA origami nanostructures such as 6-helix bundle and tetrahedral, where the nanostructures remain intact even after the covalent fixation of macromonomer layer. Presence of azide moiety in the coated DNA nanostructures were confirmed by a ‘click reaction’ using an alkyne appended model dye system.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Proof-of-concept study for the stabilization and functionalization of DNA-nanostructures using ‘template adhesion’ of guanidinium ion (Gu+) based ‘molecular glue’ was planned for the first year of the project, and the first synthesized glue monomer indicated the successful adhesion of glue monomer into a DNA bundle structure. However, solubility of the conjugate in the reaction buffer was poor. To tackle the issue, we designed a new monomer, which we could synthesize easily in two weeks. As we expected the newly synthesized monomer worked properly including an enhanced solubility of the monomer/DNA conjugate. Furthermore, we have previously experienced the covalent fixation mediated by photoirradiation as well the click reaction to DNA containing systems, hence we could conduct the planned research smoothly without specific hurdles. Additionally, all the DNA nanostructures tested (6 helix DNA bundle or tetrahedral) was synthesized by our collaborator that allowed us to focus more on the experimental parts required to achieve the research goal
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| 今後の研究の推進方策 |
For the second year, we plan to functionalize desired targeting ligand, such as RGD peptide, to the glue modified DNA nanostructure. For this purpose, our collaborator already synthesized an alkyne functionalized RGD peptide and several other peptides are under preparation. We have already confirmed the presence of azide after modifying with glue monomer by using a alkyne appended dye via copper free click reaction. After peptide conjugation, next step is to check cellular uptake of functionalized DNA nanostructure using live cells such as Hep3B and HeLa that are expressed with integrin receptor for peptide ligand recognition. Several optimizations in terms of ligand density as well as the availability of receptors on the cells are essential. Stability in extracellular matrix, fate of the DNA conjugate after cellular uptake etc need to be investigated. Then a GFP incorporated tetrahedral origami will be used to check the intracellular delivery of protein. At the end, we will test the delivery of a therapeutic enzyme using DNA tetrahedra origami structures and track its behavior inside the cells, such as endosomal escape, release of enzymes etc.
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| 次年度使用額が生じた理由 |
As the research project supported by the current funding is currently underway and further detailed study is essential to achieve the research goal. Incurring amount for the next fiscal year will be used to buy the material for research purpose, travel for attending seminars/workshops/conferences. Since next year research plan include biological cell based studies, a part of the budget will be used to buy disposable plastic items such as culture flasks, cell culturing medium, growth factors etc.
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