Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference
Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference
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DOI：
10.1089/nat.2016.0612
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发表时间：
2016-10-01
影响因子：
4
通讯作者：
Walton, S. Patrick
中科院分区：
文献类型：
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作者：
Angart, Phillip A.;Carlson, Rebecca J.;Walton, S. Patrick
有效的短干扰 RNA (siRNA) 介导的基因沉默需要选择与预期靶标互补的序列，并具有促进与 RNA 干扰 (RNAi) 途径蛋白有利的功能相互作用的序列和结构特征。在这项研究中，我们研究了 siRNA 的末端序列和结构特征如何影响 siRNA 链加载和沉默活性，以及这些特征如何最终在培养的 HeLa 细胞中产生功能不对称的双链体。我们的结果重申，确定 siRNA 活性的最重要特征是 5 末端核苷酸同一性。我们的研究结果进一步表明，siRNA 加载主要由每条 siRNA 链 5 个末端（核苷酸：1-2）的杂交稳定性控制，与相对末端无关。发现加载后，RNA 诱导的沉默复合物 (RISC) 特异性活性通过加载的 siRNA 链 5 末端（核苷酸：3-4）的较低杂交稳定性和 3 末端（核苷酸：17-18）的较高杂交稳定性得到改善。与此同时，人 Argonaute 2 (Ago2) 对 5 末端核苷酸序列的特异性识别延长了 RISC 半衰期。这些发现表明，仔细选择 siRNA 序列可以最大限度地提高预期引导链的负载量和比活性。
Efficient short interfering RNA (siRNA)-mediated gene silencing requires selection of a sequence that is complementary to the intended target and possesses sequence and structural features that encourage favorable functional interactions with the RNA interference (RNAi) pathway proteins. In this study, we investigated how terminal sequence and structural characteristics of siRNAs contribute to siRNA strand loading and silencing activity and how these characteristics ultimately result in a functionally asymmetric duplex in cultured HeLa cells. Our results reiterate that the most important characteristic in determining siRNA activity is the 5 terminal nucleotide identity. Our findings further suggest that siRNA loading is controlled principally by the hybridization stability of the 5 terminus (Nucleotides: 1-2) of each siRNA strand, independent of the opposing terminus. Postloading, RNA-induced silencing complex (RISC)-specific activity was found to be improved by lower hybridization stability in the 5 terminus (Nucleotides: 3-4) of the loaded siRNA strand and greater hybridization stability toward the 3 terminus (Nucleotides: 17-18). Concomitantly, specific recognition of the 5 terminal nucleotide sequence by human Argonaute 2 (Ago2) improves RISC half-life. These findings indicate that careful selection of siRNA sequences can maximize both the loading and the specific activity of the intended guide strand.