Minor-groove-modulating adenosine replacements control protein binding and RNAi activity in siRNAs.
Minor-groove-modulating adenosine replacements control protein binding and RNAi activity in siRNAs.
复制标题
DOI：
10.1021/cb100245u
复制
发表时间：
2010-12-17
影响因子：
4
通讯作者：
Beal, Peter A.
中科院分区：
文献类型：
--
作者：
Peacock, Hayden;Fostvedt, Erik;Beal, Peter A.
短干扰rna （sirna）是分子生物学中常见的工具，然而基于rna的治疗方法的发展受到脱靶rna结合蛋白介导的免疫刺激和非特异性作用的限制。PKR和ADAR1是参与RNAi脱靶效应的两个蛋白，它们通过双链RNA结合基序（dsRBMs）与sirna相互作用。在这里，我们报道了N2-丙炔- 2-氨基嘌呤在sirna中特异位点的引入，并随后通过铜催化叠氮化物炔环加成（CuAAC）与Nazidoacetyl-mannosamine叠氮化物或n -乙基胡椒啶叠氮化物转化为两种大体积产物。我们观察到对乘客链和引导链进行修饰对RNAi活性的位置特异性影响。根据最近对rna诱导沉默复合体（RISC）和RISC负载复合体（RLC）组成部分的结构研究，这些发现是合理的。检测了最活跃的sirna与RNA依赖性蛋白激酶（PKR）和作用于RNA 1的腺苷脱氨酶（ADAR1）的结合亲和力。无论大小如何，PKR的结合都因多次修饰而显著降低，ADAR1的结合以位置和大小敏感的方式降低。我们的研究结果提出了一种设计sirna的策略，该策略可以减少脱靶dsrbm -蛋白结合，同时保持天然RNAi活性。
Short-interfering RNAs (siRNAs) are common tools in molecular biology, however the development of RNAi-based therapeutics is limited by immunostimulatory and non-specific effects mediated by off-target RNA-binding proteins. PKR and ADAR1 are two proteins implicated in RNAi off-target effects, and share a common means of interaction with siRNAs through double-stranded RNA binding motifs (dsRBMs). Here we report the site-specific introduction of N2- propargyl 2-aminopurine into siRNAs and subsequent conversion to two bulky products via copper-catalyzed azide alkyne cycloaddition (CuAAC) with either Nazidoacetyl-mannosamine azide or N-ethylpiperidine azide. We observed position-specific effects on RNAi activity for modifications made to both the passenger and guide strands. These findings are rationalized in light of recent structural studies of components of the RNA-induced silencing complex (RISC) and RISC-loading complex (RLC). The most active siRNAs were assayed for binding affinity to the RNA-dependent protein kinase (PKR) and adenosine deaminase that acts on RNA 1 (ADAR1). PKR binding was significantly reduced by multiple modifications, regardless of size, and ADAR1 binding was reduced in a position and size-sensitive manner. Our findings present a strategy for designing siRNAs that reduce off-target dsRBM-protein binding while retaining native RNAi activity.
登录
查看更多内容
影响因子：
11.4
作者：
Heale, Bret S. E.;Keegan, Liam P.;McGurk, Leeanne;Michlewski, Gracjan;Brindle, James;Stanton, Chloe M.;Caceres, Javier F.;O'Connell, Mary A.
通讯作者：
O'Connell, Mary A.
影响因子：
46.9
作者：
Jackson, AL;Bartz, SR;Linsley, PS
通讯作者：
Linsley, PS
影响因子：
46.9
作者：
Judge, AD;Sood, V;MacLachlan, I
通讯作者：
MacLachlan, I
影响因子：
64.8
作者：
Chendrimada, TP;Gregory, RI;Shiekhattar, R
通讯作者：
Shiekhattar, R
影响因子：
2.9
作者：
Braasch, DA;Jensen, S;Corey, DR
通讯作者：
Corey, DR