Genetic code restoration by artificial RNA editing of Ochre stop codon with ADAR1 deaminase
Genetic code restoration by artificial RNA editing of Ochre stop codon with ADAR1 deaminase
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DOI：
10.1093/protein/gzz005
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发表时间：
2018-12-01
影响因子：
2.4
通讯作者：
Tsukahara, Toshifumi
中科院分区：
文献类型：
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作者：
Bhakta, Sonali;Azad, Md Thoufic Anam;Tsukahara, Toshifumi
定点诱变是重新编码遗传信息的一种非常有效的方法。将作用于 RNA 的 RNA 编辑酶腺苷脱氨酶 (ADAR) 的催化结构域与反义引导 RNA 正确连接，可以将特定的腺苷 (As) 转化为肌苷 (Is)，后者在翻译过程中被识别为鸟苷 (Gs)。人们已经努力改造 ADAR1 的脱氨酶结构域和 MS2 系统，以靶向特定的 A 残基来恢复 G -> A 突变。靶标由赭色 (TAA) 终止密码子组成，该终止密码子由编码增强型绿色荧光蛋白 (EGFP) 的氨基酸 58 (Trp) 的 TGG 密码子生成。该系统能够将终止密码子 (TAA) 转换为可读密码子 (TGG)，从而恢复细胞系统中的荧光，如 JuLi 荧光和 LSM 共聚焦显微镜所示。聚合酶链反应-限制性片段长度多态性证实了编辑的特异性，因为恢复的EGFP mRNA可以被切割成160和100个碱基对的片段。使用正义和反义引物的直接测序分析表明，5'A 的恢复率高于 3'A 的恢复率。该系统对于治疗由 G -> A 点突变引起的遗传性疾病可能非常有用。成功的体内RNA人工编辑可以加速该领域的研究，并开创针对各种遗传疾病的遗传密码恢复疗法，包括终止密码子通读疗法。
Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G -> A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5' than for the 3'A. This system may be very useful for treating genetic diseases that result from G -> A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.