Wang Debao (1918-2002) was a native of Taixing, Jiangsu Province. Wang was given a master's degree by Washington University in 1949, doctorate by Western Reserve University in 1951, and post-doctorate from US Johns Hopkins University in 1954. He was elected as an academician of Chinese Academy of Sciences in 1980. Wang is a well-known biochemical scientist. He discovered cytidine and desaminase of deoxycytidine, nucloside hydrolase of adenosine, cytidine, and xanthosine, uracil oxidase, and dephosphorylized Coenzyme A phosphokinase; He solved the problem of location of the third phosphate group in Coenzyme A; he was the first one to synthesize NADP directly from NAD in large scale, which have been applied by pharmaceutical factories all the world for 30 years; He was the first one to engage in research on biochemical process of nucleic acid, and was the founder of nucleotide fresh-making agent; Starting from 1982, Wang and his group were engaged in study of structure and function of a series of tRNA, converting yeast alanine tRNA into three types of synthetic tRNA analogues, and they prepared antibody specific to 1-methylic hypoxanthine riboside, which proved anticodon loop of tRNA is not necessarily needed in mutual identification of tRNA and aminoacyl tRNA synthetic enzyme. This work won Natural Science Award, Second Class, awarded by Chinese Academy of Sciences. Synthetic yeast alanine tRNA has all modified nucleotide and complete biologos. In 1961, he had given a biochemistry training class at a national level for the first time and composed China's first lecture file on nucleic acid: "Structure, Function and Synthesis of Nucleic Acid". Under his leadership, total synthesis of yeast alanine tRNA was completed, which won CAS Significant Science and Technology Award, First Class (1984), and State Natural Science Awards, First Class (1987).

TOTAL SYNTHESIS OF YEAST ALANINE TRANSFER RIBONUCLEIC ACID

Wang Debao

(Shanghai Institute of Biochemistry, Chinese Academy of Sciences)

Abstract

Yeast alanine transfer ribonucleic acid (tRNA^_y(Ala)) consists of 76 nucleotides. In addition to the 4common nucleotides, A, G, C and U, this tRNA contains 9 modified nucleotides of 7 different species, m¹G, D, m²₂G, I, m¹I, and T. By a combination of chemical and enzymatic methods, small oligonucleotides with lengths varying from 2 to 8 nucleotides were synthesized from mononucleotides and nucleosides. The small oligonucleotides were then phosphorylated at the 5'-end with adenosine triphosphate (ATP) in the presence of polynucleotide kinase and the 5'-phosphorylated oligonucleotides were used as donors. Donors and suitable acceptors, oligonucleotides with 3'- free hydroxyl group, were ligated by T4 RNA ligase into 6 large oligonucleotides of 13, 9, 13, 10, 12 and 19 nucleotides in lengths. These oligonucleotides were further phosphorylated at their 5'-ends and ligated to form two half molecules with 35 and 41 nucleotides respectively. Finally, the two half molicules were again phosphorylated at their 5'-ends, annealed and ligated to obtain the whole molecules of tRNAy(Ala). Prior to this, two semisyntheses 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.

Both the semi-synthetic tRNA^_y(Ala) and the synthetic tRNA^_y(Ala) occupy the same position as the natural tRNA^_y(Ala) after electrophoresis on a 20% polyacrylamide gel. They have the same chemical composition (containing 9modified nucleotides of 7different species) and structure as the natural tRNA^_y(Ala) and are biologically active, i.e. accepting and transferring alanine into proteins in a cell-free protein synthesizing system, the accepting activity of the synthetic product is 52-66%of that of the natural tRNAy(Ala) and 91-106% of that of the reconstituted product of the two natural half molecules. The incorporation activity of alanine into proteins of the synthetic 3H-alanine tRNA^_y(Ala) is 63%, corresponding to 91% of that of the natural tRNA^_y(Ala) and 115%of that of the reconstituted product of the two natural half molecules.

Both common and modified nucleotides and nucleosides, protecting and condensing reagents for chemical synthesis and enzymes for enzymatic synthesis and biological activity tests were all prepared.

To the best of our knowledge, this is the first time that a natural RNA with biological activity is synthesized.