Rational Design of RNA Editing Guide Strands: Cytidine Analogs at the Orphan Position.
Rational Design of RNA Editing Guide Strands: Cytidine Analogs at the Orphan Position.
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DOI：
10.1021/jacs.0c13319
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发表时间：
2021-05-12
影响因子：
15
通讯作者：
Beal PA
中科院分区：
文献类型：
--
作者：
Doherty EE;Wilcox XE;van Sint Fiet L;Kemmel C;Turunen JJ;Klein B;Tantillo DJ;Fisher AJ;Beal PA
作用于RNA的腺苷脱氨酶（ADARs）将双链RNA中的腺苷转化为肌苷。人类ADARs可以通过互补的引导链定向到转录组中的预定目标位点，从而允许在RNA水平上纠正致病突变。在这里，我们利用ADAR2- rna复合物的结构信息来指导核苷类似物的设计，这些核苷类似物位于引导链上与ADARs中保守的谷氨酸残基（人类ADAR2中的E488）接触的位置，该位置将腺苷翻转到ADAR活性位点进行脱胺。将该残基突变为谷氨酰胺（E488Q）可获得更高的活性，这是因为Q488具有向引导链上孤胞苷的N3提供氢键的能力。我们描述了对胞苷类似物的评价，这些类似物稳定了酶- rna复合物的激活构象，并增加了野生型酶脱氨的催化速率。ADAR2与含有胞苷类似物的双工RNA结合的新晶体结构揭示了E488与胞苷之间的密切接触，与胞苷相比，E488通过额外的氢键稳定并改变了电荷分布。在人类细胞和小鼠原代肝成纤维细胞中，与其他相同的引导寡核苷酸相比，这种单核苷酸修饰增加了定向编辑的产量。我们的研究结果表明，修饰引导RNA可以模拟过度活跃突变体的效果，并推进了招募内源性ADARs进行位点定向RNA编辑的方法。
Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
5.1
作者：
Azad, Md T. A.;Bhakta, S.;Tsukahara, T.
通讯作者：
Tsukahara, T.
影响因子：
15
作者：
Haudenschild, BL;Maydanovych, O;Beal, PA
通讯作者：
Beal, PA
影响因子：
48
作者：
Katrekar, Dhruva;Chen, Genghao;Mali, Prashant
通讯作者：
Mali, Prashant