Dynamics of co-transcriptional pre-mRNA folding influences the induction of dystrophin exon skipping by antisense oligonucleotides.
Bottom Line: In our analysis, to approximate transcription elongation, 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 structures 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.
Affiliation: Bioinformatics Institute, Singapore, Singapore.
Antisense oligonucleotides (AONs) mediated exon skipping offers potential therapy 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 splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, 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 structures 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 localisation 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 nucleotides 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 nucleotides 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.
Related in: MedlinePlus
Mentions: To demonstrate the correlative power of the fourth level scores, three common examples in which only the fourth level scores can differentiate (++) AONs in Set A (Figure 6) are discussed. Figure 6A illustrated an example wherein AON target sites with identical accessibility scores (L1) can have strikingly different engaged scores (L3). Whereas the (−) AON target site high engaged score was expected, the higher engaged score of the (++) AON target site compared to the (+) AON target site was confounding. In contrast to the L3 score, fourth level analysis showed more engaged nucleotides at the ends of (+) AON target site than at (++) AON target site, i.e. was able to discriminate between (+) and (++) AON target sites. Figure 6B illustrated an example in which the target site engaged scores (L3) correlate inversely with AON efficacy and efficiency, i.e., AON target sites with higher engaged scores had better ability to induce exon skipping. Again, the fourth level scores resolved this conundrum in a similar manner as the first example. The final example (Figure 6C) illustrated a widespread phenomenon in the data set in which (+) AON target sites had higher engaged scores (L3) than (−) AON target sites. In fact, this phenomenon caused the p-values of K-S tests of (++) vs. (+) AON target site L3 scores to be smaller than for (++) vs. (−) target site L3 scores. In most instances, most of the engaged nucleotides manifested in (+) AON target sites were localized away from the ends of the sites. Altogether, these examples showed that localization is as important as the frequency of engaged nucleotides.