Chemical Expansion of the Central Dogma towards Synthetic Microorganisms

本研究计划旨在扩展构成蛋白质生物合成系统的氨基酸、核酸和蛋白质等分子，以合成具有人工功能的新型蛋白质。最终的目标是创造出能与21种氨基酸共存的合成微生物。在这五年中，我们取得了以下成果。(1)通过使用与tRNA互补的肽核酸（PNA），一些非天然氨基酸被成功地加载到特定的tRNA上。在大肠杆菌体外蛋白质生物合成系统中进行PNA辅助的氨酰化，以产生掺入非天然氨基添加物的蛋白质。（EF-Tu）进行了扩展，将大尺寸的非天然氨基添加到大肠杆菌核糖体中，随后形成蛋白质。(3)用氨酰tRNA合成酶（ARS）和tRNA的正交组合筛选出一组能将某些非天然氨基酸引入活的哺乳动物细胞蛋白质中的酶。(4)最后，这些扩展的分子系统被引入哺乳动物细胞，并检测到含有非天然氨基酸的蛋白质的合成，尽管其数量非常少。去年，人们试图应用这些技术将荧光氨基酸连接到肽和蛋白质上。筛选荧光标记的肽以使那些与癌细胞或癌细胞特异性蛋白质结合的肽变暗。

This research project aims to expand molecules like amino acids, nucleic acids and proteins constitute the protein biosynthesizing system to synthesize novel type of proteins with artificial functions. The final goal is to create synthetic microorganisms that live with 21 type of amino acids. During the 5 year we obtained the following results. (1) Some nonnatural amino acids were successfully charged onto a specific tRNA by the use of a peptide nucleic acid (PNA) that is complementary to the tRNA. The PNA-assisted aminoacylation mold be carried out in an E.coli in vitro protein biosynthesizing system to create a protein incorporated with the nonnatural amino add. (2) tRNAs and an elongation faint Tu. (EF-Tu) were expanded to bring large-sized nonnatural amino adds into E.col ribosome and subsequently to a protein. (3) An orthogonal set of aminoacyl tRNAsynthetase (ARS) and tRNA was screened to bring some nonnatural amino acids into proteins in living mammalian cells. (4) Finally these expanded molecular systems were introduced into mammalian cells and the synthesis of proteins containing nonnatural amino acids was detected, although its quantity was very small.In the last year, an attempt was made to apply these techniques to attach fluorescent amino acids to peptides and proteins. The fluorescently labeled peptides were screened to dimmer those that bind to cancer cells or to cancer-cell specific proteins.