| 研究実績の概要 |
DNA minicircles exist in biological contexts, such as kinetoplast DNA, and are promising components for creating functional nanodevices. They have been used to mimic the topological features of nucleosomal DNA and to probe DNA-protein interactions such as HIV-1 and PFV integrases, and DNA gyrase. Here in this project, we have successfully designed, assembled, and characterized three types of rotaxanes and one type of catenane inside a DNA origami frame. These minicircles are 183 bp in length, constitute six individual single-stranded DNAs that are ligated to realize duplex interlocking, and adopt temporary base pairing of single strands for interlocking. Next, we aimed to probe the DNA-protein interactions by using these interlocked structures along with other forms of DNA. For this purpose, restriction reactions were carried out on DNAs with different topologies such as free linear duplex or duplex constrained inside origami and free or topologically-interlocked minicircles. Except the free linear duplex, all tested structures were resistant to restriction digestion, indicating that the topological features of DNA, such as flexibility, curvature, and groove orientation, play a major role in DNA-protein interactions.
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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
In the proposed work, our first aim was to construct the topologically-interlocked DNA structures inside a frame-shaped DNA origami. To this end, we have successfully designed, assembled, and characterized three types of rotaxanes and one type of catenane inside a DNA origami frame. We have achieved a consistent raw yield of 30-60% of the interlocked assemblies which is promising for nanotechnological applications. These minicircles mimic the structural features of nucleosomal DNA. Therefore, our reaction system is successfully developed. Our second aim is to analyze the DNA-protein interactions by using the DNA structure that mimics the nucleosomal DNA. For this purpose, restriction enzyme protection assays were carried out on DNAs with different topologies such as free linear duplex or duplex constrained inside origami and free or topologically-interlocked minicircles. Except the free linear duplex, all tested structures were resistant to restriction digestion, indicating that the topological features of DNA, such as flexibility, curvature, and groove orientation, play a major role in DNA-protein interactions. Therefore, the DNA-protein interactions are successfully investigated. Thus, we believe that the research is rather progressing smoothly.
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| 今後の研究の推進方策 |
As a next step, we intend to investigate the interactions of other proteins such as viral integrase on DNA for the process of retroviral integration. Initially, the HIV-1 integrase reaction will be studied. The other candidates are the MLV and PFV integration reactions. The effect of DNA length and its curvature on the integrase reaction will be analyzed in detail. The various structural forms such as free-linear dsDNA, dsDNA-confined inside a DNA origami, free- and topologically-interlocked minicircle DNAs into rotaxane and catenane will be tested for the integrase reaction. By considering the structural features of nucleosomal DNA, the curved and structurally rigid minicircle DNAs (both free and topologically-interlocked inside origami) are expected to exhibit enhanced efficiencies for the integration reactions. After the successful analysis of the integration reaction, the integrase catalytic inhibitors will be tested on the developed system. For this purpose, the commercial inhibitors such as raltegravir and elvitegravir will be used at first. Finally, the newly synthesized inhibitors will be screened by the developed method.
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