Joining of Synthetic Ribotrinucleotides with Defined Sequences Catalyzed by T4 RNA Ligase

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Abstract
RNA ligase [EC 6.5.1.3] catalyzed the joining of pC-C-Ap with C-A-A in the synthesis of C-A-A-C-C-Ap, which has the sequence of the Escherichia coli .**GRAPHIC**. 3''-end. pC-C-A was also joined to C-A-A without any undesired self-polymerization. Joining of pC-C-A to various synthetic ribotriplets, such as C-C-A, A-A-A, C-C-C, U-U-U, U-A-G, C-C-G and U-U-C, was performed as well as joining to the partially substituted trimers with a photolabile o-nitrobenzyl group, C-Anbzl-A [cytidylyl-(3''-5'')-2''-O-(o-nitrobenzyl)adenylyl-3''-5'')-adenosine] and C-C-Anbzl [cytidylyl-(3''-5'')-cytidylyl-2''-O-(o-nitrobenzyl)adenosin]. The yields were C-A-A-C-C-A (69%), C-C-A-C-C-A (38%), A-A-A-C-C-A (66%), C-C-C-C-C-A (71%), U-U-U-C-C-A (50%), U-A-G-C-C-A (23%), C-C-G-C-C-A (43%) and U-U-C-C-C-A (46%). C-Anbzl-A was a slightly poorer acceptor than C-A-A and C-C-Anbzl did not serve as an acceptor. Recognition of acceptor molecules by RNA ligase is discussed in terms of affinity of oligonucleotides for the enzyme.