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We have examined the behavior of the phosphorothioate antisense Rel A (NF-kappaB p65) oligodeoxynucleotide (oligo) and related molecules. Because of the presence of a G-tetrad near its 5'terminus, this molecule is capable of forming tetraplexes and other higher order structures in a temperature and time dependent manner. The G-tetrad in the phosphodiester congener is protected from methylation by dimethylsulfate when the oligomer is 3'-phosphorylated. However, this protection is completely lost when it is 5'phosphorylated, indicating that the formation of at least some higher order structures has been blocked. In addition, we also prevented tetraplex formation by substitution of 7-deazaguanosine (7-DG) for guanosine at several positions within and outside of the tetrad. This substitution retains Watson-Crick base pair hybridization but prevents Hoogsteen base-pair interactions. When murine K-Balb cells were treated with 20microM antisense RelA oligo, complete blockade of nuclear translocation of RelA was observed. However, this effect was virtually entirely abrogated in most cases by 7-DG substitution within the tetrad, but retained when the substitution was made 3' to the tetrad. The AS RelA-induced downregulation of Sp-1 activity behaved similarly after 7-DG substitution. Thus, the parent phosphorothioate AS RelA molecule cannot be a Watson-Crick antisense agent. However, these conclusions cannot be extrapolated to other G-tetrad containing oligomers and each must be evaluated individually.

Formation of a G-tetrad and higher order structures correlates with biological activity of the RelA (NF-kappaB p65) 'antisense' oligodeoxynucleotide