Effect of Unpaired Terminal Nucleotides of Substrate RNAs on the RNA Ligation with T4 RNA Ligase

Abstract

Abstract The effect of double-helix formations and unpaired terminal nucleotides of RNA oligomer substrates on a RNA ligation with T4 RNA ligase has been investigated by using high-performance liquid chromatography (HPLC), UV absorbance, and circular dichroism (CD) measurements, and the energy calculation for the double-helix stability of RNAs. In the 1-hour reaction of single-stranded RNA acceptors, the yield of the product increased in the order of pyrimidine donors pCp>pUp and purine donors pAp>pGp. In the reaction of a double-stranded RNA acceptor, UGCGCA, the yield also increased in the same order of the donors. On the other hand, in the reaction of double-stranded RNA acceptors containing unpaired terminal nucleotides (dangling ends), pUp and pGp donors gave the largest yields of the products in the reactions of AUGCGCA and CAUGCAU acceptors, respectively. Furthermore, the yield of the product is the largest for CAUGCAU acceptor within the acceptors having A, C, G, and U dangling ends in the reaction of pGp donor. These results sugggest that the formation of the terminal base pairs of the donors and the dangling ends may affect the RNA ligation with T4 RNA ligase.