Promotion of circular RNA production via utilizing RNA binding ligands

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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.