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

(1)新型多肽核酸的合成——侧链携带核碱基的δ-氨基酸的合成。它们被偶联在一起形成具有特定核碱基序列的肽。已发现肽核酸与互补dna杂交。本会计年度对肽核酸的化学结构进行了进一步优化。分别找到了与DNA和RNA杂交的最佳构型。(2)利用肽核酸作为RNA识别试剂，将tRNA与非天然氨基酸进行非酶化氨基化反应，合成了与n端活化氨基酸（aa^*-PNA）连接的尼尔森型肽核酸（nielsen - peptide核酸，PNA）。当DNA与tRNA和PNA互补时，形成tRNA/DNA/aa-*-PNA三元杂交体。三元杂化物发生酯交换反应，将氨基酰基转移到tRNA上。该三元体系为任何trna与非天然氨基酸的氨基酰化提供了一种简单而通用的体系。(3)链霉亲和素双突变体的合成，该突变体携带一个荧光团-猝灭基团对。将两种不同的非天然氨基酸分别携带一个荧光团和一个猝灭基团，合并到一个链霉亲和素中。这两种非天然氨基酸的位置分别由CGGG和GGGC的4碱基密码子确定。突变蛋白的分子内电子从被激发的荧光团转移到猝灭团，荧光强度降低，荧光衰变缩短。(4)携带荧光侧基的新型非天然氨基酸的合成。合成了携带较大荧光侧基的非天然氨基酸，其荧光波长大于500 nm。有些被发现成功地结合到蛋白质中。

(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.