Barker R H, Metelev V, Rapaport E, Zamecnik P
Hybridon, Inc., Worcester, MA 01605, USA.
Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):514-8. doi: 10.1073/pnas.93.1.514.
We studied inhibition of growth of the malaria parasite Plasmodium falciparum in in vitro culture using antisense (AS) oligodeoxynucleotides (ODNs) against different target genes. W2 and W2mef strains of drug-resistant parasites were exposed to AS ODNs over 48 hr, and growth was determined by microscopic examination and [3H]hypoxanthine incorporation. At ODN concentrations of 1 microM, phosphorothioate (PS) ODNs inhibited growth in a target-independent manner. However, between 0.5 and 0.005 microM, ODNs against dihydrofolate reductase, dihydropteroate synthetase, ribonucleotide reductase, the schizont multigene family, and erythrocyte binding antigen EBA175 significantly inhibited growth compared with a PS AS ODN against human immunodeficiency virus, two AS ODNs containing eight mismatches, or the sense strand controls (P < 0.0001). The IC50 was approximately 0.05 microM, whereas that for non-sequence-specific controls was 15-fold higher. PS AS ODNs against DNA polymerase alpha showed less activity than that for other targets, whereas a single AS ODN against triose-phosphate isomerase did not differ significantly from controls. We conclude that at concentrations below 0.5 microM, PS AS ODNs targeted against several malarial genes significantly inhibit growth of drug-resistant parasites in a nucleotide sequence-dependent manner. This technology represents an alternative method for identifying malarial genes as potential drug targets.
我们使用针对不同靶基因的反义（AS）寡脱氧核苷酸（ODN），在体外培养中研究了恶性疟原虫疟原虫生长的抑制情况。将抗药寄生虫的W2和W2mef菌株暴露于AS ODNs 48小时以上，并通过显微镜检查和[3H]次黄嘌呤掺入来确定生长情况。在ODN浓度为1 microM时，硫代磷酸酯（PS）ODN以非靶标依赖的方式抑制生长。然而，在0.5至0.005 microM之间，与针对人类免疫缺陷病毒的PS AS ODN、两个含有八个错配的AS ODN或有义链对照相比，针对二氢叶酸还原酶、二氢蝶酸合成酶、核糖核苷酸还原酶、裂殖体多基因家族和红细胞结合抗原EBA175的ODN显著抑制了生长（P < 0.0001）。IC50约为0.05 microM，而非序列特异性对照的IC50则高15倍。针对DNA聚合酶α的PS AS ODNs活性低于其他靶标，而针对磷酸丙糖异构酶的单个AS ODN与对照无显著差异。我们得出结论，在浓度低于0.5 microM时，针对几种疟疾基因的PS AS ODNs以核苷酸序列依赖的方式显著抑制抗药寄生虫的生长。这项技术代表了一种将疟疾基因鉴定为潜在药物靶标的替代方法。
