2019 Fiscal Year Annual Research Report
Supramolecular interactions between proteins and bioadhesive nanoparticles
| Project/Area Number |
19J23043
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| Research Institution | The University of Tokyo |
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Principal Investigator |
Morishita Kiyoshi 東京大学, 工学系研究科, 特別研究員(DC1)
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| Project Period (FY) |
2019-04-25 – 2022-03-31
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| Keywords | nanoparticle / protein / DNA / supramolecular chemistry / assembly / nanomaterials |
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| Outline of Annual Research Achievements |
This year I expanded the scope of my research on gold nanoparticle (AuNP)-protein supramolecular interactions and began studying their assembly through DNA-DNA interactions. I investigated the assembly of porous networks of AuNPs and proteins. This may lead to the development of materials for drug delivery systems, cell or tissue engineering platforms or other areas of biotechnology.
For a model protein, I decided to employ GroEL, a chaperonin protein, for its characteristic barrel shape, and ATP-responsive motion. By optimizing the AuNP size, DNA sequences, and assembly conditions, I have created a series of AuNP-GroEL building blocks (termed co-monomers) with different number of GroEL which might assemble into different geometries.
My next challenge was to isolate the individual co-monomer species. For this I trialled several established techniques, such as centrifugation, ultrafiltration, size exclusion chromatography, dialysis, and electrophoresis. Through many optimization steps, I have been able to visualize the separation of the different components of the reaction mixture. Since I have not achieved complete serration of co-monomer species, this process requires further optimization.
Separately, I have assembled co-monomers using a linker DNA. I have confirmed the assembly and studied the assemblies using dynamic light scattering (DLS), TEM, confocal microscopy, and small angle X-ray scattering (SAXS). Preliminary results also indicate the ability to encapsulate a protein guest into the assembled porous structure and release it in response to heating.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Because of challenges in the previously proposed project, I shifted my research focus early in the year to focus on the supramolecular assembly of nanoparticles and proteins. I quickly developed the skills and techniques required for this project and optimized the techniques for sample preparation. In this project, the most challenging part is anticipated to be the preparation and purification of the desired co-monomer compounds. I have identified strategies that give promising results for the preparation and purification and several important factors to consider. At the same time as this optimization has been ongoing, I have also confirmed the assembly of the co-monomers into a porous network, able to load and release a guest. This gives great hope that as the purification strategy is further optimized, the quality and properties of the assembled structure will continue to improve.
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| Strategy for Future Research Activity |
I will continue work on the development of AuNP-GroEL porous networks. So far, I have established the protocol to assemble DNA-functionalized AuNPs and GroEL into discrete hybrid nanoparticle co-monomers. While a good control over the number of GroEL on in dividual AuNPs has been demonstrated, I plan to further improve this control, either through better control over assembly conditions or more efficient post-assembly separation protocols. Once this has been optimized as much as possible, I will continue my work on the assembly of co-monomers into the desired network structure. I will characterize the assembled structures with advanced TEM techniques, such as cryo-TEM or TEM tomography, SAXS, DLS, and fluorescence imaging. I will also study the guest loading and release ability of the assembled structures, using fluorescent proteins or nanoparticles. There are two possible main focuses for this work: the assembly of structurally unique hybrid networks, and possible biotechnological properties based on structural responses to physiologically-relevant conditions. These multiple directions allow for one aspect of the project to continue even if there are unexpected setbacks in another aspect.
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