| 研究実績の概要 |
We previously identified a DNA aptamer, Apt38, which showed higher selectivity and binding affinity for ADAR1 compared to other off-target proteins using in vitro binding assays. Molecular docking and HS-AFM observation of molecular binding between Apt38 and ADAR1 further demonstrated that Apt38 is an ideal candidate for PROTAC synthesis for ADAR1.
To demonstrate the utility of the identified aptamer for the synthesis of aptamer-based PROTACs for the degradation of ADAR1, we have successfully synthesized Apt-PROTAC in which Apt38 is used as a ligand for the complex recruitment of ADAR1 and conjugated with a VHL ligand for the selective recruitment of the VHL E3 ligase. We expect this strategic design to hijack the ubiquitination process, ultimately leading to the targeted degradation of ADAR1. The construction of Apt-PROTAC was achieved through a copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. In addition, we constructed the 3D models of the Apt-PROTAC conjugate, ADAR1, and VHL3 protein for 3D modeling analysis of the tertiary complex. Our initial studies using molecular docking and HS-AFM imaging suggested the binding interaction of Apt-PROTAC with ADAR1 and VHL E3 ligase at atomic and molecular dynamic levels, respectively. We have presented this work at the 46th Annual Meeting of the Molecular Biology Society of Japan, Kobe, Japan.
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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 research objectives set out in the first-year plan have been achieved, firstly due to the ease of modification of the DNA aptamer with the DBCO group. Secondly, the simple and rapid conjugation of DBCO aptamer with azide linker containing VHL ligand by copper-free click reaction. It provided a high yield of Apt-PROTAC conjugates in the reaction. In addition, I am very grateful to the cooperation of HS-AFM and molecular docking collaborators for their continuous guidance and support of my research work, and I was able to make smooth progress towards our set goals in the first year. Now we will further optimize the HS-AFM experimental conditions and proceed with the intracellular characterization of Apt-PROTAC for the degradation of ADAR1 and anti-cancer efficiency.
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| 今後の研究の推進方策 |
In the first year, we successfully synthesized the Apt-PROTAC conjugates. This year we will continue with the following research plans:
1. To test the reliability of the 3D atomic models of Apt-PROTAC-bound ADAR1 and VHL proteins by HS-AFM observations. We plan to observe only ADAR1 and complexes with Apt-PROTAC, the VHL3 ligase complex alone, and complexes with Apt-PROTAC. Finally, Apt-PROTAC will be observed in ternary complex formation by incubation with both ADAR1 and VHL3 ligase.
2. To characterize the efficiency and mechanism of Apt38-PROTAC for the precise degradation of endogenous ADAR1 in different cancer cell lines.
3. To evaluate the anti-cancer potential of Apt-PROTAC concerning ADAR1 degradation.
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