2002 Fiscal Year Final Research Report Summary
Extension of Protein Biosynthesizing System and Incorporation of Nonnatural Amino Acids into Proteins for Chemical Extension of Protein Functions
| Project/Area Number |
11102003
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| Research Category |
Grant-in-Aid for Specially Promoted Research
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| Allocation Type | Single-year Grants |
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| Review Section |
Chemistry
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| Research Institution | Okayama University |
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Principal Investigator |
SISIDO Masahiko Okayama University, Graduate School of Natural Science and Technology, Professor, 大学院・自然科学研究科, 教授 (60026268)
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| Co-Investigator(Kenkyū-buntansha) |
HOHSAKA Takahiro Okayama University, Department of Bioscience and Biotechnology, Assistant Professor, 工学部, 助手 (30263619)
SHINOHARA Hiroaki Okayama University, Department of Bioscience and Biotechnology, Associate Professor, 工学部, 助教授 (60178887)
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| Project Period (FY) |
1999 – 2002
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| Keywords | Nonnatural Amino Acids / Peptide Nucleic Acids / Aminoacylation / 4-Base codons / Nonnatural Mutagenesis / Streptavidin / Fluorescent Amino Acids |
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| Research Abstract |
(1)Synthesis of new types of peptide nucleic acids
δ-Amino acids that carry nucleobases on their side chain have been synthesized. They were coupled together to form peptidewith a specific sequence of nucleobases. The peptide nucleic acids have been found to hybridize with complementary DNAs. In this fiscal year, further optimization of chemical structure of peptide nucleic acids was carried out. The optimum configuration for hybridization with DNA and that for hybridization with RNA were found, respectively.
(2)Nonenzymatic aminoacylation of tRNA with nonnatural amino acid by using peptide nucleic acid as an RNA recognizing agent
Nielsen-type peptide nucleic acid (PNA) linked with an activated amino acid at the N-terminal (aa^*-PNA) was synthesized. A ternary hybrid of tRNA/DNA/aa-*-PNA was formed when the DNA is complementary to the tRNA and to the PNA. An esterexchange reaction took place in the ternary hybrid to transfer the aminoacyl group to the tRNA. The ternary system provides a simple and versatilesystem for aminoacylation of any tRNAs with a nonnatural amino acid.
(3)Synthesis of double mutant of streptavidin that carry a fluorophore-quencher pair
Two different nonnatural amino acids that carry a fluorophore and a quencher, respectively, were incorporated into single streptavidin. The positions of the two nonnatural amino acids were assigned by CGGG and GGGC 4-base codons, respectively. Intramolecular electron transfer from the excited fluorophore to the quencher was observed for the mutant protein as the decrease of fluorescence intensity and as the shortening of fluorescecne decay.
(4)Synthesis of novel nonnatural amino acids that carry fluorescent side groups
Nonnatural amino acids that carry large fluorescent side groups that emit fluorescence at longer wavelengths than 500 nm were synthesized. Some were found to be successfully incorporated into proteins.
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