Chemical Expansion of the Central Dogma towards Synthetic Microorganisms

本研究项目旨在拓展氨基酸、核酸和蛋白质等构成蛋白质生物合成系统的分子，合成具有人工功能的新型蛋白质。最终目标是创造出与21种氨基酸共存的合成微生物。在5年的时间里，我们取得了以下成果。(1)利用与tRNA互补的多肽核酸(PNA)成功地将一些非天然氨基酸带入到特定的tRNA上。PNA辅助的氨基酰化模式可以在大肠杆菌的体外蛋白质生物合成系统中进行，以产生与非天然氨基酸ADD结合的蛋白质。(2)tRNA和延伸率弱于Tu。(EF-Tu)被扩展以将大尺寸的非天然氨基酸添加到E.Col核糖体中，随后形成一种蛋白质。(3)筛选了一组正交组的氨酰tRNA合成酶(ARS)和tRNA，将一些非天然氨基酸带入活哺乳动物细胞的蛋白质中。(4)将这些扩展的分子系统引入哺乳动物细胞，检测到含有非天然氨基酸的蛋白质的合成，但数量很少。去年，人们尝试将这些技术应用于将荧光氨基酸固定在多肽和蛋白质上。荧光标记的多肽被筛选出来，以使那些与癌细胞或癌细胞特异性蛋白结合的多肽变暗。

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.