Effects of Oligonucleotide Length, Mismatches and mRNA Levels on C-5 Propyne-Modified Antisense Potency

To understand the parameters required for designing potent and specific antisense C-5 propynylpyrimidine-2′-deoxyphosphorothioate-modified oligonucleotides (C-5 propyne ONs), we have utilized a HeLa line that stably expresses luciferase under tight control of a tetracycline-responsive promoter. Using this sensitive and regulatable cell-based system we have identified five distinct antisense ONs targeting luciferase and have investigated the role that ON length, target mismatches, compound stability and intracellular RNA levels play in affecting antisense potency. We demonstrate that C-5 propyne ONs as short as 11 bases retained 66% of the potency demonstrated by the parent 15 base compound, that a one base internal mismatch between the antisense ON and the luciferase 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 luciferase mRNA over 20-fold, we show that the potency of C-5 propyne ONs is unaffected by changes in the expression level of the target RNA. These data suggest that low and high copy messages can be targeted with equivalent potency using C-5 propyne ONs.