Dagle J M, Weeks D L, Walder J A
Department of Biochemistry, University of Iowa, Iowa City 52242.
Antisense Res Dev. 1991 Spring;1(1):11-20. doi: 10.1089/ard.1991.1.11.
Recently, we described a new class of antisense oligonucleotides that can be used to direct the cleavage of mRNAs in Xenopus laevis embryos by RNase H (Dagle et al., Nucleic Acids Res. 18, 4751-4757). In this study, we have examined several factors that determine the activity of these derivatives. In embryos, oligodeoxyribonucleotides were found to be rapidly degraded by a 3' exonuclease. Modification of 3'-terminal phosphodiester linkages as phosphoramidates blocks this activity. The predominant sites of endonucleolytic cleavage within the embryo are localized close to the 5' termini demonstrating the necessity of multiply modifying phosphodiester linkages at each end of the molecule. A stretch of at least six consecutive phosphodiester linkages is required to form an effective substrate for Xenopus RNase H; mRNA degradation with an oligonucleotide containing fewer than six contiguous unmodified internucleoside linkages is greatly diminished. Injection of an anti-cyclin B oligonucleotide containing eight unmodified residues results in degradation of cyclin B mRNAs and subsequent inhibition of embryonic cell division. An oligonucleotide with the same sequence but containing four consecutive phosphodiesters has no observable effect on the cell cycle. This last observation suggests that, in Xenopus embryos, hybridization alone has a limited role, if any, in oligonucleotide-mediated inhibition of gene expression.
最近，我们描述了一类新型反义寡核苷酸，其可用于在非洲爪蟾胚胎中通过核糖核酸酶H引导mRNA的切割（达格尔等人，《核酸研究》18卷，4751 - 4757页）。在本研究中，我们考察了决定这些衍生物活性的几个因素。在胚胎中，发现寡脱氧核糖核苷酸会被一种3'外切核酸酶迅速降解。将3'-末端磷酸二酯键修饰为氨基磷酸酯可阻断这种活性。胚胎内切核酸酶切割的主要位点位于靠近5'末端处，这表明有必要在分子的两端对磷酸二酯键进行多重修饰。至少一段连续六个磷酸二酯键是形成非洲爪蟾核糖核酸酶H有效底物所必需的；用含有少于六个连续未修饰核苷间连接的寡核苷酸进行的mRNA降解会大大减少。注射含有八个未修饰残基的抗细胞周期蛋白B寡核苷酸会导致细胞周期蛋白B mRNA的降解以及随后胚胎细胞分裂的抑制。具有相同序列但含有四个连续磷酸二酯键的寡核苷酸对细胞周期没有可观察到的影响。最后这一观察结果表明，在非洲爪蟾胚胎中，单独的杂交在寡核苷酸介导的基因表达抑制中作用有限（如果有作用的话）。
