A hexanucleotide pC-A-A-C-C-Ap, which corresponds to the 3'-end of tRNAfMet, was synthesized as a bisphosphorylated form, which should be a suitable donor substrate for joining with RNA ligase. 3'-Phosphorylated oligonucleotides were condensed by a triester method and the 5'-terminus of the protected hexanucleotide was phosphorylated with dianilidophosphorochloridate after demonomethoxytritylation. A site-directed base transition mutant sequence C-G-A-C-C-Ap was also synthesized by the triester method using suitably protected 3'-phosphorylated oligonucleotides.