| 研究実績の概要 |
The original goal of the project is to produce a membraneless organelle composed exclusively of DNA structures which can undergo phase separation. This DNA structure has two main components. The first one is a DNA origami particle and the second one is a DNA hydrogel host.
During the development of the project, two new technologies were developed, and their results are being organized for publication. The first of these technologies is a method for purifying DNA nano/microstructures, and the second is a method for fabricating monodisperse DNA hydrogel particles.
The purification of DNA structures was based on a pre-existing method of purification of nucleic acids called aqueous-aqueous two-phase (ATPS)fractionation, which separates molecules based on their affinity for a phase of an aqueous-aqueous emulsion. This purification method, however, had never been tested for self-assembled DNA structures. I verified that this purification method maintains the integrity of the DNA structures and it has a higher yield compared to commonly used purification methods for low volume, low concentration samples. For generating monodisperse DNA hydrogels, I developed a microfluidic device containing a microwell array, which enabled the creation of monodisperse triple-aqueous emulsions. Both methods originated from the main goal of the project of creating an artificial membraneless organelles that can undergo phase separation composed of DNA origami and DNA hydrogel.
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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
The main goal of producing an artificial membraneless organelle was not yet achieved. During the beginning of the pandemic, the projected experiments were interrupted. In this interval, computer simulations of the proposed system
were implemented, and small-scale results were obtained. After the reopening of the university, the project was resumed, producing the new techniques for preparation of DNA origami and DNA hydrogel.
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| 今後の研究の推進方策 |
In this fiscal year, five steps towards the development of an artificial membraneless organelle will be conducted: (i) I will test the incorporation and compatibility of DNA origami in DNA hydrogels developed in the previous fiscal year; (ii) I will measure and characterize the localization of DNA origami in DNA hydrogel based on the signal stimuli that induces phase separation; (iii) I will develop the theory that describes the phase separation of DNA structures based and develop simulations based on this theory; (iv) I will test the in vitro chemical activity of the artificial membraneless organelles produced and (v) I will evaluate the possibility of transfecting the artificial membraneless organelles into cells or adding them to the cell culture. I expect step (i-ii) will display a novel phenomenon of phase separation of DNA structures, (ii-iv-v) will provide evidence weather this technology will have applications in molecular biology and medicine, while (iii) will provide new insights into the phenomenon of phase separation of DNA structures that will have application in the field of genome structure.
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