Hexamer Palindromic Oligonucleotides with 5'-CG-3' Motif(s) Induce Production of Interferon

We have shown previously that 30-mer oligonucleotides containing hexamer palindromic sequences with 5'-CG-3' motif(s) induce interferon (IFN), activate natural killer (NK) cells, and thus exhibit tumor-regressing activity. The present study showed that a hexamer palindromic oligonucleotide (5'-AACGTT-3') alone induced IFN from mouse spleen cells when added with cationic liposomes. Accordingly, 32 kinds of hexamer palindromic oligonucleotides were tested for their ability to induce IFN in the presence of cationic liposomes. The results show that oligonucleotides with NACGTN and NTCGAN sequences exhibited the strongest activity. ACGCGT and TCGCGA also possessed moderate but significant activity. In contrast, palindromes without CG motif(s) were devoid of the activity. No hexamer oligonucleotides showed the activity when liposomes were absent. A complete palindromic sequence was essential as any single base substitution resulted in diminished activity. Among variety of palindromic oligonucleotides of different sizes with an ACGT sequence at the center, the tetramer oligonucleotide was without activity, whereas the activity of hexamer and longer oligonucleotides was almost equally high. These results strongly suggest that the minimal essential structure required for IFN induction is the hexamer palindromic sequence with CG motif(s).