Department of Chemistry, University of California, Davis, CA, USA.
Department of Molecular and Cellular Biology, University of California, Davis, CA, USA.
Nucleic Acids Res. 2024 Jul 8;52(12):6733-6747. doi: 10.1093/nar/gkae461.
Adenosine Deaminases Acting on RNA (ADARs) are enzymes that catalyze the conversion of adenosine to inosine in RNA duplexes. These enzymes can be harnessed to correct disease-causing G-to-A mutations in the transcriptome because inosine is translated as guanosine. Guide RNAs (gRNAs) can be used to direct the ADAR reaction to specific sites. Chemical modification of ADAR guide strands is required to facilitate delivery, increase metabolic stability, and increase the efficiency and selectivity of the editing reaction. Here, we show the ADAR reaction is highly sensitive to ribose modifications (e.g. 4'-C-methylation and Locked Nucleic Acid (LNA) substitution) at specific positions within the guide strand. Our studies were enabled by the synthesis of RNA containing a new, ribose-modified nucleoside analog (4'-C-methyladenosine). Importantly, the ADAR reaction is potently inhibited by LNA or 4'-C-methylation at different positions in the ADAR guide. While LNA at guide strand positions -1 and -2 block the ADAR reaction, 4'-C-methylation only inhibits at the -2 position. These effects are rationalized using high-resolution structures of ADAR-RNA complexes. This work sheds additional light on the mechanism of ADAR deamination and aids in the design of highly selective ADAR guide strands for therapeutic editing using chemically modified RNA.
腺苷脱氨酶作用于 RNA（ADARs）是一种酶，可催化 RNA 双链中腺苷向肌苷的转化。这些酶可用于纠正转录组中导致疾病的 G 到 A 突变，因为肌苷被翻译为鸟苷。向导 RNA（gRNA）可用于指导 ADAR 反应到特定的位点。需要对 ADAR 向导链进行化学修饰，以促进递送、增加代谢稳定性，并提高编辑反应的效率和选择性。在这里，我们表明 ADAR 反应对向导链中特定位置的核糖修饰（例如 4'-C-甲基化和锁核酸（LNA）取代）非常敏感。我们的研究得益于含有新的核糖修饰核苷类似物（4'-C-甲基腺苷）的 RNA 的合成。重要的是，LNA 或 ADAR 向导中的不同位置的 4'-C-甲基化强烈抑制 ADAR 反应。虽然 LNA 在向导链位置-1 和-2 处阻断 ADAR 反应，但 4'-C-甲基化仅在-2 位抑制。使用 ADAR-RNA 复合物的高分辨率结构可以合理地解释这些效应。这项工作进一步阐明了 ADAR 脱氨酶的作用机制，并有助于设计用于化学修饰 RNA 的治疗性编辑的高度选择性 ADAR 向导链。
