Kean J M, Kipp S A, Miller P S, Kulka M, Aurelian L
Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Biochemistry. 1995 Nov 14;34(45):14617-20. doi: 10.1021/bi00045a001.
Antisense oligonucleoside methylphosphonates complementary to the 12 nucleotides found at the intron/exon junction of the splice acceptor site of herpes simplex virus type 1 (HSV-1) immediate early mRNAs 4 and 5 were synthesized. The methylphosphonate oligomers contained either 2'-deoxyribose nucleosides, d-OMPs, or 2'O-methylribose nucleosides, mr-OMPs. At 37 degrees C, the affinity of the mr-OMP for a complementary 12-mer RNA target was approximately four times higher than that of the corresponding d-OMP as measured by a constant activity gel electrophoresis mobility shift assay. An mr-OMP whose sequence contained two mismatched bases did not bind to the RNA target under these conditions. The mr-OMP also showed improved ability to inhibit HSV-1 replication in HSV-1 infected Vero cells in culture. Thus the IC50 of the mr-OMP was five times less than that of the d-OMP. No inhibition was observed by the mismatched mr-OMP, and no inhibition of herpes simplex virus type 2 (HSV-2) replication was observed with any of the oligomers. These results demonstrate a direct correlation between oligomer binding affinity and antisense activity in cell culture and suggest that oligo-2'-O-methylribonucleoside methylphosphonates are promising candidates for development of effective antisense reagents.
合成了与单纯疱疹病毒1型（HSV-1）立即早期mRNA 4和5的剪接受体位点的内含子/外显子交界处发现的12个核苷酸互补的反义寡核苷酸甲基膦酸酯。甲基膦酸酯寡聚物包含2'-脱氧核糖核苷（d-OMP）或2'-O-甲基核糖核苷（mr-OMP）。在37℃下，通过恒定活性凝胶电泳迁移率变动分析测定，mr-OMP对互补的12聚体RNA靶标的亲和力比相应的d-OMP高约四倍。在这些条件下，其序列包含两个错配碱基的mr-OMP不与RNA靶标结合。mr-OMP在培养的HSV-1感染的Vero细胞中还显示出改善的抑制HSV-1复制的能力。因此，mr-OMP的IC50比d-OMP低五倍。错配的mr-OMP未观察到抑制作用，并且任何寡聚物均未观察到对单纯疱疹病毒2型（HSV-2）复制的抑制作用。这些结果证明了细胞培养中寡聚物结合亲和力与反义活性之间的直接相关性，并表明寡聚2'-O-甲基核糖核苷甲基膦酸酯是开发有效反义试剂的有前途的候选物。
