2017 Fiscal Year Annual Research Report
Studying the moecular basis of Hepatitis C virus replication with Atomic Force Microscory
| Project/Area Number |
16F16390
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| Research Institution | Kyoto University |
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Principal Investigator |
野田 岳志 京都大学, ウイルス・再生医科学研究所, 教授 (00422410)
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| Co-Investigator(Kenkyū-buntansha) |
GILMORE JAMIE 京都大学, ウイルス・再生医科学研究所, 外国人特別研究員
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| Project Period (FY) |
2016-11-07 – 2019-03-31
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| Keywords | RNA / atomic force microscopy |
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| Outline of Annual Research Achievements |
Over the past year, I have developed models for RNA structural domains by correlating AFM data with secondary structure models predicted from RNAstructure software. After developing these models, I synthesized minimal RNA constructs favoring particular conformations of each domain. AFM imaging of these constructs helped to validate the models, in addition to providing a way to independently evaluate the properties of each domain. From the AFM-based structural models of domains in viral RNA, I further proposed mutations that are likely to disrupt these structures. With the help of one of the students in the Noda lab, Junichi Kajikawa, we are working to introduce these mutations into viral RNA. These experiments should help to further validate the structural models as well as to identify the functional role of these domains in the viral lifecycle. I am currently preparing a manuscript to report these findings. While last year’s publication provided proof-of-concept for this technology by comparing AFM data of RNA molecules to RNA molecules with known structural domains (rRNA), this manuscript will be the first to actually demonstrate the use of AFM for identification and modeling of RNA structure in molecules with a previously unknown structure.
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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
Currently, I am working on a manuscript which will be the first to demonstrate the use of AFM to model the structure of RNA molecules. Thus, I believe that I have successfully established AFM as a new technology for structural analysis of RNA molecules which represents a breakthrough for the field of RNA biology. Furthermore, I have identified new structural domains in viral RNA molecules which could serve as antiviral targets in the future
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| Strategy for Future Research Activity |
Although some domains have been successfully modeled and verified using RNAstructure, the structure of some of the other domains is less certain. I hope to perform experiments to refine and validate the precise structure of these domains. I have a variety of ideas for how to approach this issue. First, I want to attempt to use single particle reconstruction techniques, such as those normally applied for cryo-EM data, to improve the resolution of the AFM images. In addition, since models generated thus far using RNAstructure are only 2D representations of the data, the correlation between these models and AFM data is not completely clear. I have begun using modeling platforms like 3dRNA-2.0 to generate 3D representations of the data. I want to further extend this analysis by trying to overlay the 3D models with my AFM data. This would allow our models to further account for topological information. In addition, factors favoring the formation of particular conformational states of the RNA domains can be further understood by performing thermodynamic analysis. To accomplish this, I have been in conversations with Dr. Luis Marky, a Professor who was previously on my graduate committee at my alma mater who specializes in thermodynamic analysis of nucleic acids.
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