A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects.
A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects.
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DOI：
10.1093/nar/gkq341
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发表时间：
2010-09
影响因子：
14.9
通讯作者：
Kjems J
中科院分区：
文献类型：
--
作者：
Bramsen JB;Pakula MM;Hansen TB;Bus C;Langkjær N;Odadzic D;Smicius R;Wengel SL;Chattopadhyaya J;Engels JW;Herdewijn P;Wengel J;Kjems J
小干扰RNA（siRNA）由于其固有的miRNA样行为而被确立为抑制哺乳动物细胞中基因功能的优选工具，但却引发非预期的基因沉默。这种脱靶效应主要由siRNA种子区（从5′端开始计数的任一siRNA链的位置2-8）与（脱靶）靶标的3′UTR中的互补序列之间的序列特异性相互作用介导。之前已经表明，siRNA的化学修饰可以减少脱靶，但只有非常少的修饰被测试，留下更多的有待鉴定。在这里，我们开发了一种基于荧光素酶的测定法，其适合于使用稳定的细胞系以高通量方式监测siRNA脱靶。我们使用10种不同类型的化学修饰、三种不同的靶序列和三种siRNA浓度研究了siRNA种子内化学修饰单核苷酸位置对siRNA功能和脱靶的影响。我们发现了几种不同修饰的siRNA以减少脱靶，但将强烈不稳定的解锁核酸（UNA）修饰掺入siRNA的位置7中最有效地减少了所有测试序列的脱靶。值得注意的是，siRNA-靶相互作用的这种位置特异性去稳定化没有显著降低siRNA效力，因此非常适合于未来的siRNA设计，特别是对于siRNA浓度较低的体内应用。
Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.
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影响因子：
64.8
作者：
Baek, Daehyun;Villen, Judit;Shin, Chanseok;Camargo, Fernando D.;Gygi, Steven P.;Bartel, David P.
通讯作者：
Bartel, David P.
影响因子：
46.9
作者：
Jackson, AL;Bartz, SR;Linsley, PS
通讯作者：
Linsley, PS
对所有单核苷酸不匹配的靶位点，对活性siRNA的沉默效应进行系统分析。
影响因子：
14.9
作者：
Du, Q;Thonberg, H;Wang, J;Wahlestedt, C;Liang, ZC
通讯作者：
Liang, ZC
影响因子：
14.9
作者：
Lin, XY;Ruan, X;Anderson, MG;McDowell, JA;Kroeger, PE;Fesik, SW;Shen, Y
通讯作者：
Shen, Y
影响因子：
12.4
作者：
Howard, Kenneth A.;Rahbek, Ulrik L.;Kjems, Jorgen
通讯作者：
Kjems, Jorgen