Small-Molecule Aptamer for Regulating RNA Functions in Mammalian Cells and Animals,Journal of the American Chemical Society

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Small-Molecule Aptamer for Regulating RNA Functions in Mammalian Cells and Animals
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2023-03-29 , DOI: 10.1021/jacs.2c12332
Keisuke Fukunaga ₁ , V Dhamodharan ₁ , Nao Miyahira ₁ , Yoko Nomura ₁ , Kamila Mustafina ₁ , Yasuaki Oosumi ₂ , Kosuke Takayama ₂ , Akira Kanai ₂ , Yohei Yokobayashi ₁

Affiliation

Synthetic riboswitches that can regulate gene expression by a small molecule recognized by an RNA aptamer in mammalian cells have various potential applications in biotechnology and medicine. However, the variety of small molecules and their cognate aptamers that have been demonstrated to function in mammalian cells is limited. The currently available aptamer-ligand pairs also require high small molecule concentrations to enable gene regulation, making them less desirable for industrial and biomedical applications. We conducted in vitro selection of RNA aptamers against a small molecule ASP7967 whose structure is closely related to ASP2905, a known inhibitor of potassium voltage-gated channel sub-family H member 3 (KCNH3). One of the aptamers selected (AC17–4) was found to be functional in HEK293 cells, and it was used to design aptazyme-based riboswitches that can activate gene expression (>10-fold) in the presence of ASP2905 or ASP7967 at as low as 5 μM in the culture medium. An aptazyme-based riboswitch was successfully used to regulate human erythropoietin expression in mice injected with an adeno-associated virus (AAV8) vector using orally administered ASP7967. Furthermore, by combining aptazyme-based and exon-skipping riboswitch mechanisms, an ON/OFF ratio approaching 300 was achieved with a low basal expression level in cultured cells.

中文翻译：

用于调节哺乳动物细胞和动物 RNA 功能的小分子适体

可以通过哺乳动物细胞中 RNA 适体识别的小分子调节基因表达的合成核糖开关在生物技术和医学中具有各种潜在应用。然而，已证明在哺乳动物细胞中起作用的小分子及其同源适体的种类是有限的。目前可用的适体-配体对也需要高浓度的小分子来实现基因调控，这使得它们不太适合工业和生物医学应用。我们在体外进行针对小分子 ASP7967 选择 RNA 适体，其结构与 ASP2905 密切相关，ASP2905 是已知的钾电压门控通道亚家族 H 成员 3 (KCNH3) 抑制剂。发现所选的适体之一 (AC17–4) 在 HEK293 细胞中具有功能，它被用于设计基于适体酶的核糖开关，在 ASP2905 或 ASP7967 存在的情况下，以低至在培养基中为 5 μM。基于 aptazyme 的核糖开关成功地用于调节注射腺相关病毒 (AAV8) 载体的小鼠体内促红细胞生成素的表达，该载体使用口服给药的 ASP7967。此外，通过结合基于适体酶和外显子跳跃的核糖开关机制，在培养细胞中以低基础表达水平实现了接近 300 的开/关比。

更新日期：2023-03-29

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