Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61--77 from Escherichia coli tRNAfMet.
Chemically synthesized fragments corresponding to the 3' end of tRNAfMet from Escherichia coli were joined by T4-induced RNA ligase to yield a heptadecanucleotide (bases 61--77). The 3' terminus of C-C-A was modified by introduction of the ethoxymethylidene group to prevent intra- and intermolecular self-joining reactions at the 3' end. The terminal trimer was phosphorylated using polynucleotide kinase and joined to C-A-A with RNA ligase. The hexamer [C-A-A-C-C-A(ethoxymethylidene)] corresponding to bases 72--77 was obtained in a yield of 60%. An undecanucleotide (bases 61--71) which had been synthesized in a yield of 34% by similar enzymatic joining of U-C-C-G-G to pC-C-C-C-C-G was allowed to react with the 5'-phosphorylated hexamer (bases 72--77) using an excess of RNA ligase to yield the heptadecanucleotide U-C-C-G-G-C-C-C-C-C-G-C-A-A-C-C-A (bases 61--77). The product was identified by homochromatography and nearest neighbor analysis.
D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism; D009239:N-Formylmethionine; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000096:biosynthesis*; D009843:Oligoribonucleotides / Q000096:biosynthesis*; D011118:Polynucleotide Ligases / Q000378:metabolism*; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D012343:RNA, Transfer / Q000096:biosynthesis*; D012260:Ribonucleases
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D009239:N-Formylmethionine; D012343:RNA, Transfer; D012260:Ribonucleases; D011118:Polynucleotide Ligases; D011130:RNA Ligase (ATP)