Dynamics of co-transcriptional pre-mRNA folding influences the induction of dystrophin exon skipping by antisense oligonucleotides

Abstract

Antisense oligonucleotide (AONs) mediated exon skipping offers potential theraphy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before slicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elonagtion, a "window of analysis" that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structure were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered "engaged" if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localization of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency) of 94% of 176 previously reported AONs. Four novel insights are inferred: (1) the lowest frequencies of engaged nucleotide are associated with the most efficient AONs; (2) engaged nucleotides at 3′ or 5′ ends of the target site attenuate AON performance more than at other sites; (3) the performance of longer AONs is less attenuated by engaged nucleotide at 3′ or 5′ ends of the target site compared to shorter AONs; (4) engaged nucleotides at 3′ end of a short target site attenuates AON efficiency more than at 5′ end. © 2008 Wee et al.