Promotion of circular RNA production via utilizing RNA binding ligands

在此之前，我展示了rna结合小分子萘啶氨基甲酸二聚体（NCD）在细胞环境中上调circRNA产生的作用，使用了一种产生已知circRNA的模型pre-mRNA: circZKSCNAN1。其中，当NCD结合位点存在于反向互补序列rcs中时，NCD稳定了内含子间相互作用。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（ONs）上调circRNA的可行性。为了实现这一目标，设计了桥接on，该桥接on针对环状外显子两侧内含子中的两个远端序列的组合，并在物理上桥接两侧内含子。这种概念的适用性在用质粒转染的HeLa细胞中进行了测试，以表达以前用于NCD的模型pre-mRNA。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，该寡核苷酸都上调了细胞中circZKSCAN1的产生，上调幅度高达3.4倍。而对照on则没有出现上调。说明桥接相互作用的形成及其对ON生物活性的重要性。利用原生PAGE、ITC和SPR进一步确定桥接相互作用和结合亲和力的形成。总的来说，我能够证明ON在调节circRNA产生方面的能力。

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.