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The substrate specificity of T4 RNA ligase has been examined to determine whether the intermolecular reaction is sufficiently general to realize its potential in the enzymatic synthesis of oligoribonucleotides of defined sequence. Reactions between a variety of acceptor molecules with 3'- and 5'-bydroxyl groups and donor molecules with 3'- and 5'-phosphates indicate that the minimal substrates are a trinucleoside diphosphate acceptor and a nucleoside 3',5'-bisphosphate donor. Increasing the chain length of either the acceptor or donnor has little effect on the rate or extent of reaction. Although the base composition of the donor has only a small effect on the reaction rate, the presence of uridine in the acceptor greatly reduces the amount of product formed. The presence of a phosphate on the 3' terminus of the donor molecule permits a unique intermolecular product with a 5'-hydroxyl and a 3'-phosphate. By enzymatically either adding a 5'-phosphate or removing the 3'-phosphate, a new donor or acceptor is prepared so synthesis of an oligomer chain can proceed in either direction. With the simplicity of this enzymatic pathway and the rather broad substrate specificity of T4 RNA ligase, a convenient method for the synthesis of oligoribonucleotides is established.

A simple procedure is described to prepare nucleoside 3'(2'),5'-bisphosphates from the corresponding nucleosides with the use of pyrophosphoryl chloride. This method is rapid, gives nearly quantitative yields and, most importantly, can be used for a variety of nucleosides with base and sugar modifications. Since 3',5'-bisphosphates are donors in the T4 RNA ligase reaction, a single residue can be enzymatically attached to the 3' end of oligoribonucleotides. By these procedures, five different ring-modified nucleosides and one sugar-modified nucleoside were incorporated onto the 3' end of (Ap)3C. In two cases, an additional step of synthesis with RNA ligase resulted in the modified nucleotide being located in an internal position in the oligonucleotide. Thus, a general method for the synthesis of oligoribonucleotides containing modified nucleosides is outlined. Since many of the modified nucleosides are fluorescent, oligomers containing them should be useful in a variety of physical and biochemical studies.

RNA ligase isolated from bacteriophage T4-infected Escherichia coli will utilize a number of different compounds with the general structure Ado-5'PP-X as substrates in an ATP-independent reaction. The P-X portions of these molecules are transferred to the 3'-hydroxyl of an oligoribonucleotide to form a phosphodiester bond, and the Ado-5'P (AMP) portion is released. AMP (4,5,7,8). The formation of the phosphodiester bond between the two enzyme-bound oligomers presumably occurs via the nucleophilic attack of the 3'-hydroxyl group of the acceptor on the activated 5'-phosphoryl group of the donor with the elimination of AMP. Because the adenylylated donor can react with the acceptor in the absence of ATP (4,5,8), it is likely to be an intermediate of the reaction, as is the case with T4 DNA ligase (9). This last step in the RNA ligase reaction between an adenylylated donor oligoribonucleotide (Ado-5'PP5'-RNA) and an acceptor oligoribonucleotide is the major topic of this paper. We have examined a number of analogous pyrophosphates with the general structure Ado-5'PP-X for their ability to serve as donors in a similar ATP-independent reaction in which the P-X moiety is transferred to the acceptor (Ap)3C. We find that many members of this simple class of compounds exclusively donate their nonadenylyl groups to an oligoribonucleotide acceptor. AMP is the other product of the reaction. The structural variability of the groups added and the yields of products obtained suggest that this reaction of RNA ligase will be a useful method for the addition of a wide variety of compounds to the 3' terminus of RNA molecules.