Muhonen Pirkko, Tennilä Tuula, Azhayeva Elena, Parthasarathy Ranga N, Janckila Anthony J, Väänänen H Kalervo, Azhayev Alex, Laitala-Leinonen Tiina
Bone Biology Research Consortium, Institute of Biomedicine, Department of Anatomy, University of Turku.
Chem Biodivers. 2007 May;4(5):858-73. doi: 10.1002/cbdv.200790073.
Short interfering RNA (siRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA-induced silencing complex RISC, which requires degradation of the sense strand of the siRNA duplex. Although various chemical modifications have been introduced to the antisense strand, modifications to the Argonaute2 (Ago2) cleavage site in the sense strand have, so far, not been described in detail. In this work, novel 2'-F-purine modifications were introduced to siRNAs, and their biological efficacies were tested in cells stably expressing human tartrate-resistant acid phosphatase (TRACP). A validated siRNA that contains both purine and pyrimidine nucleotides at the putative Ago2 cleavage site was chemically modified to contain all possible combinations of 2'-fluorinated 2'-deoxypurines and/or 2'-deoxypyrimidines in the antisense and/or sense strands. The capacity of 2'-F-modified siRNAs to knock down their target mRNA and protein was studied, together with monitoring siRNA toxicity. All 2'-F-modified siRNAs resulted in target knockdown at nanomolar concentrations, despite their high thermal stability. These experiments provide the first evidence that RISC activation not only allows 2'-F modifications at the sense-strand cleavage site, but also increase the biological efficacy of modified siRNAs in vitro.
基于RNA干扰（RNAi）的药物开发需要具有良好基因沉默特性的短干扰RNA（siRNA）分子。RNAi的第一步是激活RNA诱导沉默复合体RISC，这需要降解siRNA双链体的正义链。尽管已对反义链进行了各种化学修饰，但迄今为止，尚未详细描述对正义链中Argonaute2（Ago2）切割位点的修饰。在这项研究中，将新型2'-F-嘌呤修饰引入siRNA，并在稳定表达人抗酒石酸酸性磷酸酶（TRACP）的细胞中测试其生物学效应。在假定的Ago2切割位点同时含有嘌呤和嘧啶核苷酸的经过验证的siRNA经过化学修饰，使其反义链和/或正义链中含有2'-氟化2'-脱氧嘌呤和/或2'-脱氧嘧啶的所有可能组合。研究了2'-F修饰的siRNA敲低其靶mRNA和蛋白质的能力，并监测了siRNA的毒性。尽管2'-F修饰的siRNA具有高热稳定性，但所有这些siRNA在纳摩尔浓度下均能导致靶标敲低。这些实验首次证明，RISC激活不仅允许在正义链切割位点进行2'-F修饰，还能提高修饰后的siRNA在体外的生物学效应。
