Petrova Kruglova Natalya S, Meschaninova Mariya I, Venyaminova Alya G, Zenkova Marina A, Vlassov Valentin V, Chernolovskaya Elena L
Laboratory of Nucleic Acids Biochemistry, Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia.
Oligonucleotides. 2010 Dec;20(6):297-308. doi: 10.1089/oli.2010.0246. Epub 2010 Oct 28.
The thermodynamic asymmetry of siRNA duplexes determines their silencing activity. Favorable asymmetry can be achieved by incorporation of mismatches into the 3' part of the sense strand, providing fork-siRNAs, which exhibit higher silencing activity and higher sensitivity to nucleases. Recently, we found that selective 2'-O-methyl modifications of the nuclease-sensitive sites of siRNA significantly improve its nuclease resistance without substantial loss of silencing activity. Here, we examined the impact of nucleotide mismatches and the number and location of 2'-O-methyl modifications on the silencing activity and nuclease resistance of anti-MDR1 siRNAs. We found that both nonmodified and selectively modified fork-siRNAs with 4 mismatches at the 3' end of the sense strand suppress the expression of target gene at lower effective concentrations than the parent siRNAs with classical duplex design. The selective modification of nuclease-sensitive sites significantly improved the stability of fork-siRNAs in the presence of serum. The selectively modified fork-siRNA duplexes provided inhibitory effect over a period of 12 days posttransfection, whereas the gene silencing activity of the nonmodified analogs expired within 6 days. Thus, selective chemical modifications and structural alteration of siRNA duplexes improve their silencing properties and significantly prolong the duration of their silencing effect.
小干扰RNA（siRNA）双链体的热力学不对称性决定了它们的沉默活性。通过在正义链的3'部分引入错配可实现有利的不对称性，从而产生叉状siRNA，其具有更高的沉默活性和对核酸酶更高的敏感性。最近，我们发现对siRNA核酸酶敏感位点进行选择性2'-O-甲基修饰可显著提高其核酸酶抗性，而不会大幅丧失沉默活性。在此，我们研究了核苷酸错配以及2'-O-甲基修饰的数量和位置对抗多药耐药蛋白1（MDR1）siRNAs沉默活性和核酸酶抗性的影响。我们发现，与具有经典双链设计的亲本siRNAs相比，在正义链3'端带有4个错配的未修饰和选择性修饰的叉状siRNAs在更低的有效浓度下就能抑制靶基因的表达。在血清存在下，对核酸酶敏感位点的选择性修饰显著提高了叉状siRNAs的稳定性。选择性修饰的叉状siRNA双链体在转染后12天内都具有抑制作用，而未修饰类似物的基因沉默活性在6天内就消失了。因此，siRNA双链体的选择性化学修饰和结构改变改善了它们的沉默特性，并显著延长了其沉默效果的持续时间。
