2015 Fiscal Year Annual Research Report
| Project/Area Number |
15F15333
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| Research Institution | The University of Tokyo |
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Principal Investigator |
菅 裕明 東京大学, 理学(系)研究科(研究院), 教授 (00361668)
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| Co-Investigator(Kenkyū-buntansha) |
OBEXER RICHARD 東京大学, 理学(系)研究科(研究院), 外国人特別研究員
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| Project Period (FY) |
2015-11-09 – 2018-03-31
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| Keywords | ペプチド / ケミカルバイオロジー / 翻訳 |
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| Outline of Annual Research Achievements |
A model lipid bilayer with an area of roughly 0.5 mm x 0.5 mm was constructed. A high fraction of phospholipids had to be used in order to achieve stability. The sheer size of this lipid bilayer however, causes it to be very fragile towards convections within the aqueous drop. In parallel, a new puromycin linker derivative was synthesized for mRNA display. Rather than using PEG, poly-N-methyl-aminocaproic acids that are functionalized to be coupled to DNA and puromycin were constducted. This linker is designed to span across the lipid bilayer, maintaining the link between geno- and pheno-type.The RaPID selection against small reactive probes was also executed. Next generation sequencing of isolated sequences revealed convergence, indicating a successful outcome of the selection. These peptides will be useful tools for protein labeling and protein immobilization in cell biology and biotechnology.
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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
Almost all parts for the new selection assay for identifying cell permeable peptides are ready to be tested. So far, most steps were successful or were helpful to identify better alternatives. In the case of the model lipid bilayer, for instance, the performed experiments showed, that using multiple small model lipid bilayers will be superior to using a single large, with regard to stability as well as experimental robustness.
At this point, the selection of peptides against small molecular probes seems to have been successful. The obtained results indicate that the selected peptides could react with a broad range of activated small molecules, such as alkyl halides. Therefore, these peptides are interesting candidates for a broad range of potential applications, ranging from catalytic peptides to biohybrid materials.Overall, it seems that the achieved results by Dr. Obexer to date is very satisfactory and the experiments were progressing according to the original plan.
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| Strategy for Future Research Activity |
In the next step the new puromycin linker for mRNA display will be prepared and the membrane permeability will be tested. In parallel a chip for supported model membranes will be tested, previously designed by Watanabe et al.. Using this chip, the lipid mixtures with a composition that is more reminiscent of natural cell membranes will be tested. Hereafter all parts of the assay will be assembled. Using a well-studied model target, the experimental feasibility of the developed assay for selecting cell permeable assay will be demonstrated. If successful, we will perform a selection against a relevant intracellular target, such as influenza RNA polymerase.In parallel, the reactive peptides will be kinetically analyzed and their substrate scope probed. In vivo labeling within cells will demonstrate their utility for protein tracking. Protein immobilization on surfaces will be tested with regard to protein function and surface concentration.
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