Retroviral integration into topologically-interlocked DNAs to probe the role of DNA structure and screen viral inhibitors
| Project/Area Number |
21K05274
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Multi-year Fund |
|---|
| Section | 一般 |
|---|
| Review Section |
Basic Section 37010:Bio-related chemistry
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
A. RAJENDRAN 京都大学, エネルギー理工学研究所, 講師 (90723122)
|
|---|
| Project Period (FY) |
2021-04-01 – 2024-03-31
|
| Project Status |
Granted (Fiscal Year 2022)
|
| Budget Amount *help |
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2023: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
Fiscal Year 2022: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2021: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
|
|---|
| Keywords | Topological structures / DNA nanotechnology / Minicircle DNA / DNA-protein interactions / Atomic force microscopy / DNA origami / Interlocked structures / DNA minicircles / HS-AFM / Viral inhibitor / Viral integrase / Drug screening |
|---|
| Outline of Research at the Start |
The topologically-interlocked minicircle DNAs that mimic the structural features of nucleosomal DNA will be prepared inside a frame-shaped DNA origami. The structures will then be used as substrates to probe the role of DNA structure on retroviral integration. The efficiency and selectivity of integration will be evaluated on various DNA forms. Finally, the acquired knowledge will be used to develop and screen the integrase catalytic inhibitors by using the interlocked DNA substrates.
|
| Outline of Annual Research Achievements |
Topology-specific DNA structures, such as small loops, supercoils, knots, and catenated mini and maxicircles, occur in numerous instances in vivo. Besides their presence in vivo, synthetic molecules with interlocked units were achieved in macromolecular chemistry decades ago and are the current topic of interest in structural DNA nanotechnology. Due to the circular nature of these minicircles and interlocked DNAs, their structure and topology resemble nucleosomal DNA. Therefore, DNA minicircles with and without interlocked structures are attractive targets to probe the topological features of DNA and DNA-protein interactions. In this study, we aimed to design and prepare the DNA minicircles in free and topologically interlocked forms. These minicircles mimic nucleosomal DNA in terms of size and structure. Also, we have designed a frame-shaped DNA origami nanostructure and incorporated the topologically interlocked rotaxanes and catenanes. During our study, we noticed that these minicircles/interlocked structures and also the DNA origami frame are relatively unstable under application-specific conditions. Thus, before the investigation of the DNA-protein interactions and drug screening, it was necessary to stabilize these structures. We spent a reasonable amount of time in developing the stabilization methods for these DNA structures, and finally achieved stable substrates for the DNA-protein interactions and drug screening.
|
| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In this study, our first objective was to design and prepare the DNA minicircles that mimic nucleosomal DNA. Once we prepared these DNA minicircles, we planned to incorporate these minicircles inside a frame-shaped DNA origami nanostructure in a topologically-interlocked pattern. As planned, we have successfully designed and prepared the DNA minicircles. Also, the topologically-interlocked DNA structures inside the DNA origami frame were incorporated. The reaction conditions were optimized so as to obtain a high yield of the interlocked nanostructures. During the course of this study, we evaluated the stability of these nanostructures under biological conditions. We found that the synthesized nanostructural assemblies are relatively unstable and are not suitable for the intended characterization, such as DNA-protein interactions and inhibitor screening. Then we aimed in developing stabilization methods that can help the topologically-interlocked assembly suitable for DNA-protein interactions. At present, we have successfully developed methods to stabilize the nanostructures under biological environments. Thus, we believe that this research is rather progressing smoothly.
|
| Strategy for Future Research Activity |
After the successful synthesis of the DNA minicircles and interlocked structures inside the origami frame, we plan to investigate their stability under protein buffers, cell lysate, and other biological conditions. Once the required parameters and conditions are optimized, the interactions of the proteins such as viral integrase on DNA for the process of retroviral integration will be investigated. Initially, the effect of DNA length and its structure on the integrase reaction will be studied in detail. The various structural forms such as linear duplex DNA, DNA-confined inside a DNA origami, free- and topologically-interlocked minicircle DNAs into rotaxane and catenane will be tested for the integrase reaction. This will offer us the effect of structural features on the DNA-protein interactions as in the case of nucleosome. Once the role of DNA structure in the integration reaction is known, the integrase catalytic inhibitors (both commercially available and newly synthesized) will be tested on the synthesized DNA nanostructures. This study will be extended to several other proteins and their inhibitors as well.
|
Report
(2 results)
Research Products
(8 results)
