Thermodynamic basis of selectivity in guide-target-mismatched RNA interference.
Thermodynamic basis of selectivity in guide-target-mismatched RNA interference.
复制标题
引导-靶标-错配 RNA 干扰选择性的热力学基础。
DOI：
10.1002/prot.24025
复制
发表时间：
2012-05
影响因子：
2.9
通讯作者：
Osman, Roman
中科院分区：
文献类型：
--
作者：
Joseph, Thomas T.;Osman, Roman
RNAi中的沉默受到引导链/靶mRNA错配的强烈影响。靶成核被认为发生在引导物的位置2-8（“种子区”）;该区域中的成功杂交是靶结合亲和力的主要决定因素，因此是靶切割的主要决定因素。为了确定RNAi中靶序列选择性的分子基础，我们研究了种子区7个位置中所有可能的不同单一错配-总共21个取代。我们报告了软核热力学积分模拟的结果，以确定由于引导链中的单个错配而导致的Argonaute的靶结合自由能的变化，该错配在不完全匹配的靶mRNA的结合过程中出现。与实验一致，大多数错配损害靶结合，与RNAi序列选择性中结合亲和力变化的突出作用一致。发现位于错配碱基对附近的单个Argonaute残基对结合亲和力的变化有显著贡献。我们还采用这种方法来分析Argonaute的DNA·RNA杂交体解离的错配依赖性自由能变化，作为miRNAs从沉默途径逃逸的模型。miRNA的几个错配序列增加了对Argonaute的亲和力，这意味着一些错配可能会降低逃逸的概率。此外，结合ssDNA的碱基取代依赖性自由能变化的计算揭示了如引导链与Argonaute结合所预期的温和的序列敏感性。我们的研究结果为RNAi靶序列选择性提供了热力学基础，并表明miRNA错配可能会增加沉默效果，因此可能在进化上具有优势。
Silencing in RNAi is strongly affected by guide-strand/target-mRNA mismatches. Target nucleation is thought to occur at positions 2–8 of the guide (“seed region”); successful hybridization in this region is the primary determinant of target binding affinity and hence target cleavage. To define a molecular basis for the target sequence selectivity in RNAi, we studied all possible distinct single mismatches in seven positions of the seed region — a total of 21 substitutions. We report results from soft-core thermodynamic integration simulations to determine changes in target binding free energies to Argonaute due to single mismatches in the guide strand, which arise during binding of an imperfectly matched target mRNA. In agreement with experiment, most mismatches impair target binding, consistent with a prominent role for binding affinity changes in RNAi sequence selectivity. Individual Argonaute residues located near the mismatched base pair are found to contribute significantly to binding affinity changes. We also employ this methodology to analyze the mismatch-dependent free energy changes for dissociation of a DNA·RNA hybrid from Argonaute, as a model for the escape of miRNAs from the silencing pathway. Several mismatched sequences of the miRNA have increased affinity to Argonaute, implying that some mismatches may reduce the probability for escape. Furthermore, calculations of base-substitution-dependent free energy changes for binding ssDNA reveal mild sequence sensitivity as expected for guide strand binding to Argonaute. Our findings give a thermodynamic basis for RNAi target sequence selectivity and suggest that miRNA mismatches may increase silencing effectiveness and thus could be evolutionarily advantageous.
登录
查看更多内容
影响因子：
2.8
作者：
BEUTLER, TC;MARK, AE;VANGUNSTEREN, WF
通讯作者：
VANGUNSTEREN, WF
影响因子：
4.8
作者：
Lima, Walt F.;Wu, Hongjiang;Crooke, Stanley T.
通讯作者：
Crooke, Stanley T.
影响因子：
16.8
作者：
Haley, B;Zamore, PD
通讯作者：
Zamore, PD
影响因子：
4.4
作者：
JORGENSEN, WL;CHANDRASEKHAR, J;KLEIN, ML
通讯作者：
KLEIN, ML
影响因子：
5.6
作者：
Draper, DE
通讯作者：
Draper, DE