T4-Induced RNA Ligase Joins Single-Stranded Oligoribonucleotides

RNA ligase isolated from Escherichia coli infected with bacteriophage T4 will catalyze the formation of an intermolecular 3′→ 5′phosphodiester linkage between an oligoribonucleotide with a free 3′-hydroxyl and another oligoribonucleotide with a 5′-phosphate. Upon reaction with (Ap)5C, nearly quantitative conversion of the hexamer [5′-32P]p(Up)5U to the dodecamer (Ap)5C[3′→ 5′-32P]p(Up)5U was observed. The product was identified by its mobility on RPC-5 column chromatography, its resistance to alkaline phosphatase, and the appearance of the expected radiolabeled products on hydrolysis with alkali, ribonuclease A, snake venom phosphodiesterase, and spleen phosphodiesterase. The coupling of other pairs of single-stranded oligoribonucleotides has also been demonstrated. The intermolecular joining reaction is probably mechanistically similar to the intramolecular cyclization activity previously reported for T4 RNA ligase. It is expected that this enzyme will be useful for the synthesis of RNA fragments of defined sequence.