- Unexpected products detected in oligoribonucleotide synthesis reactions catalyzed by T4 RNA ligase are shown to be a result of a partial reversal of the enzyme reaction. A transfer assay for the reversal of the third step in the RNA ligase reaction mechanism and an exchange assay for the reversal of both the second and third steps are described. Reversal is confirmed by the formation of the expected covalent intermediates, adenylylated donor and adenylyl ligase, from a reaction containing 5'-AMP, unadenylylated ligase, and the tetranucleotide (Ap)3Cp. In the reverse reaction, RNA ligase shows a strong preference for hydrolysis of the 3'-terminal phosphodiester bonds of oligoribonucleotides which terminate in a 3'-phosphate. Several strategies are discussed to minimize the effects of reversal in the enzymatic synthesis of oligoribonucleotides.