Single-stranded phosphodiester and phosphorothioate oligonucleotides bind actinomycin D and interfere with tumor necrosis factor-induced lysis in the L929 cytotoxicity assay.

Antisense oligonucleotides may prove useful tools to elucidate the biological functions of cytokines and to address regulatory control of cytokine expression. The functional activity of cytokines generally is determined by biological assays, and a standard biological assay for TNF activity measures the cytotoxic effect of TNF on actinomycin D-sensitized L929 cells (Matthews and Neale, 1987). We observed that phosphorothioate and phosphodiester oligonucleotides, at concentrations > 100 nM in supernatants tested in this bioassay, prevented TNF-induced lysis of L929 cells. This "protective" effect was due to an interaction of the single-stranded oligonucleotides with actinomycin D as demonstrated by UV spectra of an actinomycin D-oligonucleotide solution. Substitution of cycloheximide for actinomycin D in the L929 assay eliminated the protective effect of the oligonucleotide. Our results reinforce the importance of controlling for nonspecific effects of oligonucleotides, particularly when a functional assay for protein activity is used to screen for antisense-mediated reductions in target protein expression.