Stull R A, Zon G, Szoka F C
Department of Pharmacy, University of California, San Francisco 94143.
Antisense Res Dev. 1993 Fall;3(3):295-300. doi: 10.1089/ard.1993.3.295.
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.
反义寡核苷酸可能是阐明细胞因子生物学功能以及解决细胞因子表达调控问题的有用工具。细胞因子的功能活性通常通过生物学检测来确定，一种用于检测肿瘤坏死因子（TNF）活性的标准生物学检测方法是测量TNF对放线菌素D致敏的L929细胞的细胞毒性作用（马修斯和尼尔，1987年）。我们观察到，在该生物检测中所测试的上清液中，浓度>100 nM的硫代磷酸酯和磷酸二酯寡核苷酸可防止TNF诱导的L929细胞裂解。如放线菌素D - 寡核苷酸溶液的紫外光谱所示，这种“保护”作用是由于单链寡核苷酸与放线菌素D的相互作用所致。在L929检测中用环己酰亚胺替代放线菌素D消除了寡核苷酸的保护作用。我们的结果强化了控制寡核苷酸非特异性效应的重要性，特别是当使用蛋白质活性功能检测来筛选反义介导的靶蛋白表达降低时。
