2016 Fiscal Year Research-status Report
Development of new method for screening anticancer drugs that target topoisomerases by using DNA origami
| Project/Area Number |
16K17934
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| Research Institution | Kyoto University |
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Principal Investigator |
A. RAJENDRAN 京都大学, エネルギー理工学研究所, 講師 (90723122)
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| Project Period (FY) |
2016-04-01 – 2018-03-31
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| Keywords | DNA origami / Topoisomerase / DNA rotaxanes / DNA catenane / Topoisomerase inhibitors / high speed AFM |
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| Outline of Annual Research Achievements |
In this project, I have developed a novel method by the combination of DNA origami and high-speed atomic force microscopy (HS-AFM) for the screening of Topoisomerase (Topo)-inhibitors. As for the target structures for the Topo reactions, I have constructed the topologically interlocked DNA catenane- and rotaxane-like structures inside a frame-shaped DNA origami. The formation of the DNA origami frame and the insertion of the catenane- and rotaxane-like structures were successfully characterized by agarose gel electrophoresis and HS-AFM. The optimization of the conditions for the formation and insertion of the catenane/rotaxane inside the DNA origami frame were carried out. To increase the stability of these functional structures, the nicks in these structures were sealed by using T4 DNA ligase. The ligation was also confirmed by the thermal treatment of these structures, where the ligated samples were stable at high temperature incubation while the non-ligated samples failed to keep the topologically interlocked structures. Further, I have investigated the stability of the DNA origami frame and the catenane/rotaxane ring structures in the presence of various kinds of Topo inhibitors. Both the origami and the DNA ring are stable against the Topo inhibitors for several hours at room temperature. This indicated that the DNA origami based analysis of Topo inhibitors could be successfully carried out. These functional structures inside the DNA origami frame contain the binding site for Topos. The Topo reaction and the function of Topo-inhibitors are under investigation.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The project is progressing smoothly as originally planned. The first step of this project is to prepare the DNA origami and to insert the topologically interlocked nanostructures such as DNA catenane and rotaxanes. This is successfully carried out recently. The conditions for the formation of these interlocked structures were also optimized. The stability of the interlocked structures were improved by ligating the nicks in the DNA strands. This lead to the reasonably good yield of these nanostructures inside the DNA origami. Further, the DNA origami and the catenane/rotaxanes structures were found to be stable against the Topo inhibitors. This indicates that our nanoplatform can be successfully applied for the analysis of Topo reactions and their inhibitors. The second step of this project, the Topo reaction and the function of Topo-inhibitors, are under investigation which is expected to progress without any trouble. Part of these results were presented in the 97th annual meeting of the Chemical Society of Japan, held at Keio University during 16-19 March 2017.
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| Strategy for Future Research Activity |
The first step of the future plan in this project is to analyse the enzymatic reaction of Topo on the target site in the DNA rotaxane and catenane. For this purpose the unbound DNA strands and the DNA ligase should be removed before the Topo reactions. I have already planned to develop methods for the purification and quantification of the purified samples. Once this step is successfully performed, the Topo reactions will be carried out. For this purpose, the design strategies, methods, and protocols were all planned, and the necessary chemicals and reagents were ordered. The second stage of the future plan is to screen the Topo inhibitors. The methods for this screening and the experimental setups are already prepared. Some of the commercially available Topo inhibitors were purchased and recently developed inhibitors were kindly received form my collaborator. The Topo enzyme and the enzyme activity analysis kit were also purchased. Over all, the future plan is well designed, experimental setups were made, and the experiments were already started.
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| Causes of Carryover |
In the future plan, I am planning to use the Topoisomerase enzymes and the enzyme activity analysis kits. This will cost more than the previous year budget. So, I would like to use more money in the next year. Also, the I need to buy the commercially available Topoisomerase inhibitors.
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| Expenditure Plan for Carryover Budget |
In this year, the additional budget is planned to use for the purchase of Topoisomerase and DNA Ligase enzymes, and Topoisomerase inhibitors. Also, the additional money will be used for the development of the purification protocol and method to quantify the nanostructures.
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