EFFECTS OF OLIGONUCLEOTIDE LENGTH, MISMATCHES AND MESSENGER-RNA LEVELS ON C-5 PROPYNE-MODIFIED ANTISENSE POTENCY

To understand the parameters required for designing potent and specifi c antisense C-5 pynyl-pyrimidine-2'-deoxyphosphorothioate-modified oli gonucleotides (C-5 propyne ONs), we have utilized a HeLa line that sta bly expresses luciferase under tight control of a tetracycline-respons ive promoter. Using this sensitive and regulatable cell-based system w e have identified five distinct antisense ONs targeting luciferase and have investigated the role that ON length, target mismatches, compoun d stability and intracellular RNA levels play in affecting antisense p otency, We demonstrate that C-5 propyne ONs as short as 11 bases retai ned 66% of the potency demonstrated by the parent 15 base compound, th at a one base internal mismatch between the antisense ON and the lucif erase target reduced the potency of the antisense ON by 43% and two or more mismatches completely inactivated the antisense ON and that C-5 propyne ONs have a biologically active half-life in tissue culture of 35 h, In addition, by regulating the intracellular levels of the lucif erase mRNA over 20-fold, we show that the potency of C-5 propyne ONs i s unaffected by changes in the expression level of the target RNA, The se data suggest that low and high copy messages can be targeted with e quivalent potency using C-5 propyne ONs.