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Extension of Protein Biosynthesizing System and Incorporation of Nonnatural Amino Acids into Proteins for Chemical Extension of Protein Functions

蛋白质生物合成系统的扩展以及将非天然氨基酸掺入蛋白质中以化学扩展蛋白质功能

基本信息

批准号：
11102003
负责人：
SISIDO Masahiko
金额：
$ 108.86万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Specially Promoted Research
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2002
项目状态：
已结题

项目摘要

(1)Synthesis of new types of peptide nucleic acidsδ-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 agentNielsen-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 pairTwo 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 groupsNonnatural 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.

（1）新型肽核酸的合成了侧链上带有核碱基的δ-氨基酸。它们偶联在一起形成具有特定序列的核碱基的肽。已发现肽核酸与互补DNA杂交。于本财政年度，进一步优化肽核酸的化学结构。分别找到了与DNA杂交和与RNA杂交的最佳构型。（2）以肽核酸为RNA识别剂，非天然氨基酸与tRNA的非酶氨酰化反应合成了N端连接有活化氨基酸的Nielsen型肽核酸（aa^*-PNA）。当DNA与tRNA和PNA互补时，形成tRNA/DNA/aa-*-PNA三元杂交体。在三元杂交体中发生酯交换反应，将氨酰基转移到tRNA上。该三元体系为任何tRNA与非天然氨基酸的氨酰化提供了一个简单而可靠的方法。（3）链霉亲和素双突变体的合成将两种不同的非天然氨基酸分别掺入到单一链霉亲和素中，所述两种非天然氨基酸分别携带荧光团和猝灭剂。这两个非天然氨基酸的位置分别由CGGG和GGGC四碱基密码子指定。突变蛋白的荧光强度降低，荧光衰减时间缩短，分子内电子从激发态荧光团转移到猝灭剂。（4）新型带荧光侧基的非天然氨基酸的合成了带大荧光侧基的非天然氨基酸，其在500 nm以上的波长处发射荧光。其中一些被成功地整合到蛋白质中。