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 T4 RNA ligase into six large oligonucleotides (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 (tRNAAlay). 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. Both the semi-synthetic tRNAAlay and the synthetic tRNAAlay occupy the same position as the natural tRNAAlay after electrophoresis on a 20% polyacrylamide gel. They have the same chemical composition (containing 9 modified nucleotides of 7 different species) and structure as the natural tRNAAlay 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 tRNAAlay 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 tRNAAlay is 63%, corresponding to 91% of that of the natural tRNAAlay and 115% of that of the reconstituted product of the two natural half molecules. To the best of our knowledge, this is the first time that a natural RNA with biological activity is synthesized.
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D012346:RNA, Transfer, Amino Acyl / Q000138:chemical synthesis* / Q000494:pharmacology; D015003:Yeasts / Q000032:analysis*
D012346:RNA, Transfer, Amino Acyl