FORMATION OF A G-TETRAD AND HIGHER-ORDER STRUCTURES CORRELATES WITH BIOLOGICAL-ACTIVITY OF THE REIA (NF-KAPPA-B P65) ANTISENSE OLIGODEOXYNUCLEOTIDE

We have examined the behavior of the phosphorothioate antisense ReI A (NF-kappa B p65) oligodeoxynucleotide (oligo) and related molecules, B ecause of the presence of a G-tetrad near its 5' terminus, this molecu le is capable of forming tetraplexes and other higher order structures in a temperature and time dependent manner, The G-tetrad in the phosp hodiester congener is protected from methylation by dimethyl-sulfate w hen the oligomer is 3'-phosphorylated. However, this protection is com pletely lost when it is 5' phosphorylated, indicating that the formati on of at least some higher order structures has been blocked, In addit ion, we also prevented tetraplex formation by substitution of 7-deazag uanosine (7-DG) for guanosine at several positions within and outside of the tetrad, This substitution retains Watson-Crick base pair hybrid ization but prevents Hoogsteen base-pair interactions, When murine K-B alb cells were;treated with 20 mu M antisense ReIA oligo, complete blo ckade of nuclear translocation of ReIA was observed, However, this eff ect was virtually entirely abrogated in most cases by 7-DG substitutio n within the tetrad, but retained when the substitution was made 3' to the tetrad, The AS ReIA-induced downregulation of Sp-l activity behav ed similarly after 7-DG substitution. Thus, the parent phosphorothioat e AS ReIA 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.