Promotion of circular RNA production via utilizing RNA binding ligands

利用 RNA 结合配体促进环状 RNA 的产生

• 批准号: 22KJ2154
• 负责人: Ni Lu
• 金额: $ 1.09万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2023
• 资助国家: 日本
• 起止时间: 2023-03-08 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22KJ2154/
• 关键词: circular RNA; back-splicing; small molecules; oligonucleotides

Previously, I demonstrated the utility of an RNA-binding small molecule, naphthyridine carbamate dimer (NCD), in upregulating the production of circRNA in a cellular environment using a model pre-mRNA that produces a known circRNA: circZKSCNAN1. Where, NCD stabilized the inter-intronic interaction when its binding site was present in the reverse complementary sequences RCSs.To extend the concept further, in this fiscal year, I explored the feasibility of utilizing oligonucleotides (ONs) for circRNA upregulation. To achieve this, a bridging-ON was designed which targets a combination of two distant sequences in the introns flanking the circularizing exon(s) and physically bridges the flanking introns. The applicability of such a concept was tested in HeLa cells transfected with plasmids to express the model pre-mRNA used previously with NCD. The RT-qPCR results show the oligonucleotide upregulating the production of circZKSCAN1 in cells regardless of the stability of the interaction between the flanking introns, achieving up to 3.4-fold upregulation. While control ONs showed no upregulation. Indicating the formation and importance of the bridging interaction for the biological activity of the ON. The formation of the bridging interaction and binding affinity was further determined using native PAGE, ITC, and SPR. Overall, I was able to demonstrate ON's capacity in modulating circRNA production.

以前，我证明了RNA结合小分子，萘啶氨基甲酸酯二聚体（NCD）的效用，在细胞环境中使用产生已知circRNA的模型前mRNA：circZKSCNAN 1上调circRNA的产生。其中，当NCD的结合位点存在于反向互补序列RCS中时，NCD稳定了内含子间的相互作用。为了进一步扩展这一概念，在本财政年度，我探索了利用寡核苷酸（ON）上调circRNA的可行性。为了实现这一点，设计了桥接-ON，其靶向环化外显子侧翼的内含子中的两个远端序列的组合，并物理桥接侧翼内含子。在用质粒转染以表达先前与NCD一起使用的模型前mRNA的HeLa细胞中测试这种概念的适用性。RT-qPCR结果显示，无论侧翼内含子之间相互作用的稳定性如何，寡核苷酸都上调细胞中circZKSCAN 1的产生，实现高达3.4倍的上调。而对照ON没有显示上调。表明桥接相互作用的形成和对ON的生物活性的重要性。使用非变性PAGE、ITC和SPR进一步确定桥接相互作用的形成和结合亲和力。总的来说，我能够证明ON调节circRNA产生的能力。

项目成果

Exploring methods in regulating circular RNA production in cellular model

探索细胞模型中调节环状RNA产生的方法

• 发表时间: 2023
• 作者: Lu Ni;Takeshi Yamada;Kazuhiko Nakatani
• 通讯作者: Kazuhiko Nakatani

Exploring new methods in regulating circular RNA production using cellular model

利用细胞模型探索调控环状RNA产生的新方法

• 发表时间: 2023
• 作者: Lu Ni;Takeshi Yamada;Kazuhiko Nakatani
• 通讯作者: Kazuhiko Nakatani