酵母丙氨酸转移核糖核酸3′-半分子(36—76)的合成

本文报告用T_4RNA连接酶将相应于酵母丙氨酸转移核糖核酸(tRNA_y~(Ala))3′-半分子(36—76)的三个寡核昔酸大片段——10(36—45)(Ⅰ),12(46—57)(Ⅱ)和19p(58—76)(Ⅲ)——从3′-端向5′-端延伸逐个连接合成了这个tRNA的3′-半分子(36—76)。首先在供受体配比为1:1.5的情况下,采取三步连续反应,即19p(Ⅲ)的5′-磷酸化,然后与12(Ⅱ)的连接和连接反应产物的5′-磷酸化等反应,一次制备分离的方法,以70％的总得率合成了5′-磷酸化的三十一核苷酸(46—76)(Ⅳ)。然后,以(Ⅳ)作为下一步反应的供体和三倍量的10(Ⅰ)连接,以67％的产率合成了具有四十一核苷酸的tRNA_y~(Ala)的3′-半分子(36—76)(Ⅴ)。将这个合成的3′-半分子,5′-磷酸化以后,与天然的5′-半分子连接,人工半合成了tRNA_y~(Ala)整分子,经生物活力测定,这个人工半合成的tRNA_y~(Ala)具有接受[~3H]-丙氨酸、并能将接受的丙氨酸转移到蛋白质分子中去的生物活力。     

酵母丙氨酸转移核糖核酸的人工全合成

本文报道采用有机合成与酶促合成相结合的方法,按照R.Holley等测定又经他人修正的一级结构,人工全合成了酵母丙氨酸转移核糖核酸(tRNA_y~(Ala))的工作。我们合成的tRNA_y~(Ala)与天然的tRNA_y~(Ala)具有相同的化学组成(含有全部修饰核苷酸)和结构,并有完整的生物活力,即在大鼠肝氨酰基tRNA合成酶的催化下,能接受丙氨酸,而且在兔网织红细胞裂解液体系中能将所携带的丙氨酸参入到蛋白质中去。在进行全合成以前,曾分别进行了两种人工半合成,即将天然的5′半分子与人工的3′半分子和人工的5′半分子与天然的3′半分子进行连接,都取得有生物活力的整分子tRNA_y~(Ala)。据我们了解,这是世界上第一次用人工方法合成的具有生物功能的核糖核酸。     

SYNTHESIS OF THE 3'-HALF MOLECULE OF YEAST ALANINE tRNA

This paper deals with the synthesis of the 3'-half molecule of yeast alanine transfer RNA (tRNA_y~(Ala)) by ligation with T_4 RNA ligase of three component oligonucleotide fragments corresponding to nucleotides 36-45(Ⅰ), 46-57(Ⅱ) and 58-76(Ⅲ) in succession extending from the 3'-end to the 5'-end. First, in a ratio of acceptor to donor at 1.5 to 1, we adopted a method of three successive reactions, namely, the 5'-phosphorylation of the nonadecamer (Ⅲ), ligation with the dodecamer(Ⅱ) and the 5'-phosphorylation of the ligation product formed; with one isolation step and obtained the 5'-phosphorylated 31mer(46-76) (Ⅳ) in an overall yield of 70%. Then the 31met(Ⅵ) as a donor was ligated with 3 times of decamer(Ⅰ) to form the 41met(36-76) (Ⅴ), the 3'-half molecule of tRNA_y~(Ala)). The yield was 67%. After 5'-phosphorylation, (Ⅴ) was ligated with the natural 5'-half molecule to form the semi-synthetic tRNA_y~(Ala)), which was biologically active, i. e. accepting and transferring (~3H)-alanine into p     

TOTAL SYNTHESIS OF YEAST ALANINE TRANSFER RIBONUCLEIC ACID

By a combination of chemical and enzymatic methods, small oligonucleotides with lengths varying from 2 to 8 nucleotides were synthesized from mononucleotides. The small oligonucleotides were then ligated with T_4 RNA ligase into six laxge ligonucleotides (9 to 19 nucleotides long) which were further ligated to form two half molecules with 35 and 41 nucleotides respectively, Finally, the two synthetic half molecules were annealed and ligated to obtain the whole molecule of yeast alanine tRNA (tRNA_y~(Ala)). Prior to this, two semi-syntheses were performed, i. e. ligation of the synthetic 5'-half molecule with the natural 3'-half molecule and that of the natural 5'-half molecule with the synthetic 3'-half molecule.