Thermodynamic control of mismatch discrimination for extensive splicing regulation of PKM pre-mRNA

In this article, we present an approach to maximizing the splicing regulatory properties of splice-switching oligonucleotide (SSO) designed to regulate alternative splicing of pyruvate kinase M (PKM) pre-mRNA. The studied SSO interacts with the regulatory element in exon 10 of PKM pre-mRNA and contributes to a significant reduction of PKM2 level with a simultaneous increase of the PKM1 isoform. This SSO forms a duplex not only with the regulatory fragment of exon 10 but also with a similar RNA fragment of intron 9. The impact of this unspecific interaction on SSO regulatory properties, as well as the functional role of the intron 9 fragment, remains debatable. Herein, two types of modified nucleotides, unlocked nucleic acids (UNAs) and locked nucleic acids (LNAs), were used to study mismatch discrimination within duplexes involving modified SSOs. Our studies showed that LNAs increased mismatch discrimination, resulting in stronger regulatory properties of SSO. On the other hand, UNA reduced mismatch discrimination, decreasing the potentially therapeutic properties of SSO. The results indicate that specific interactions with exon 10 are more favorable for therapeutic applications than simultaneous hybridization with intron 9 and exon 10. The results also suggest the lack of a regulatory role for the intron 9 target site in alternative splicing of PKM pre-mRNA.