<br>使用 8-Azanebularine 修饰的 RNA 双链体选择性抑制 ADAR1,Biochemistry

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使用 8-Azanebularine 修饰的 RNA 双链体选择性抑制 ADAR1
Biochemistry ( IF 2.9 ) Pub Date : 2023-03-27 , DOI: 10.1021/acs.biochem.2c00686
Herra G Mendoza ₁ , Victorio Jauregui Matos ₁ , SeHee Park ₁ , Kevin M Pham ₁ , Peter A Beal ₁

Affiliation

作用于 RNA 的腺苷脱氨酶（ADAR）是 RNA 编辑酶，可催化 dsRNA 中腺苷（A）水解脱氨为肌苷（I）。在人类中，两种催化活性的 ADAR ADAR1 和 ADAR2 执行这种 A-to-I 编辑事件。不断发展的核苷酸碱基编辑领域突出了 ADAR 是有前途的治疗剂，而多项研究也确定了 ADAR1 在癌症进展中的作用。然而，由于缺乏对 ADAR1 识别 RNA 的详细分子理解，定点定向 RNA 编辑的潜力以及抑制剂的合理设计受到了阻碍。在这里，我们设计了含有核苷类似物 8-氮杂杂草碱（8-azaN）的短 RNA 双链体，以深入了解人类 ADAR1 催化结构域的分子识别。通过凝胶移位和体外脱氨实验，我们验证了 ADAR1 催化结构域的双链体二级结构要求，并提出了结合的最小双链体长度（14 bp，其中 5 bp 5' 和 8 bp 3' 到编辑位点）。这些发现与 ADAR1 催化结构域先前结构模型中预测的 RNA 结合接触一致。最后，我们确定作为游离核苷的 8-azaN 和带有 8-azaN 的 ssRNA 都不抑制 ADAR1，并证明 8-azaN 修饰的 RNA 双链选择性抑制 ADAR1，而不是密切相关的 ADAR2 酶。

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Selective Inhibition of ADAR1 Using 8-Azanebularine-Modified RNA Duplexes

Adenosine deaminases acting on RNA (ADARs) are RNA editing enzymes that catalyze the hydrolytic deamination of adenosine (A) to inosine (I) in dsRNA. In humans, two catalytically active ADARs, ADAR1 and ADAR2, perform this A-to-I editing event. The growing field of nucleotide base editing has highlighted ADARs as promising therapeutic agents while multiple studies have also identified ADAR1’s role in cancer progression. However, the potential for site-directed RNA editing as well as the rational design of inhibitors is being hindered by the lack of detailed molecular understanding of RNA recognition by ADAR1. Here, we designed short RNA duplexes containing the nucleoside analog, 8-azanebularine (8-azaN), to gain insight into molecular recognition by the human ADAR1 catalytic domain. From gel shift and in vitro deamination experiments, we validate ADAR1 catalytic domain’s duplex secondary structure requirement and present a minimum duplex length for binding (14 bp, with 5 bp 5′ and 8 bp 3′ to editing site). These findings concur with predicted RNA-binding contacts from a previous structural model of the ADAR1 catalytic domain. Finally, we establish that neither 8-azaN as a free nucleoside nor a ssRNA bearing 8-azaN inhibits ADAR1 and demonstrate that the 8-azaN-modified RNA duplexes selectively inhibit ADAR1 and not the closely related ADAR2 enzyme.

更新日期：2023-03-27

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