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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.

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Antisense oligodeoxynucleotides are potential therapeutic agents, but their development is still limited by both a poor cellular uptake and a high degradation rate in biological media. The strategy that we propose to face these problems is to use small synthetic carriers, around 30 nm diameter, the SupraMolecular Bio Vectors (SMBV). We used positively charged SMBV and settled the ionic incorporation of negatively charged oligonucleotides into these carriers. A minimal leakage of 10% of total incorporated oligonucleotides was then measured during two months. Both protection and uptake of oligonucleotides were then analyzed. On the one hand, we showed that the incorporation of oligonucleotides into the selected SMBV allows to significantly increase, 8 times, their half-life, in cell growth medium. On the other hand, the internalization of the SMBV, into cells, by an endosomal pathway has been characterized. The essential point is that the SMBV uptake elicits the simultaneous oligonucleotide uptake. The oligonucleotide amount that goes through cells within 5 h can be up to 30 times higher than for free oligonucleotides and the fraction of oligonucleotides that is present in the cytosol is increased up to 10 fold after incorporation into the SMBV. This study demonstrates the ability of SMBV to improve oligonucleotide cellular behaviour.