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

Wee KB, Pramono ZA, Wang JL, MacDorman KF, Lai PS, Yee WC - PLoS ONE (2008)

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.

View Article: PubMed Central - PubMed

Affiliation: Bioinformatics Institute, Singapore, Singapore.

ABSTRACT
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.

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Occurrences of engaged nucleotides at each step of transcriptional analysis.In the above illustration, the horizontal axis denotes sequential steps of transcriptional analysis while the vertical axis denotes numbered nucleotides within the AON target site. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The calculations of the fourth level scores, L4_OR and L4_AND, are illustrated (refer to main text for details).
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pone-0001844-g003: Occurrences of engaged nucleotides at each step of transcriptional analysis.In the above illustration, the horizontal axis denotes sequential steps of transcriptional analysis while the vertical axis denotes numbered nucleotides within the AON target site. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The calculations of the fourth level scores, L4_OR and L4_AND, are illustrated (refer to main text for details).

Mentions: The nucleotide accessibility scores at the first and second levels of analysis are mean scores. As a result, two nucleotides with identical accessibility scores may have markedly different numbers of unpaired predicted secondary structures at each step of transcriptional analysis. In analyzing accessibility for AON binding, it may be important to take into account steps of transcriptional analysis in which a nucleotide is predicted to have total absence of unpaired secondary structures, i.e. the nucleotide is predicted to be completely inaccessible or “engaged” at the particular step of transcriptional analysis (Figure 2B). For the purpose of analysis, at every step of transcriptional analysis, each nucleotide in the AON target site which is engaged may then be depicted in a plot as illustrated in Figure 3. Table S3 of the Online Supporting Information tabulates these plots for all the AON target sites analyzed.


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

Wee KB, Pramono ZA, Wang JL, MacDorman KF, Lai PS, Yee WC - PLoS ONE (2008)

Occurrences of engaged nucleotides at each step of transcriptional analysis.In the above illustration, the horizontal axis denotes sequential steps of transcriptional analysis while the vertical axis denotes numbered nucleotides within the AON target site. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The calculations of the fourth level scores, L4_OR and L4_AND, are illustrated (refer to main text for details).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2267000&req=5

pone-0001844-g003: Occurrences of engaged nucleotides at each step of transcriptional analysis.In the above illustration, the horizontal axis denotes sequential steps of transcriptional analysis while the vertical axis denotes numbered nucleotides within the AON target site. At each step of transcriptional analysis, nucleotides in the target site that are engaged are depicted as a black dot in the plot. The calculations of the fourth level scores, L4_OR and L4_AND, are illustrated (refer to main text for details).
Mentions: The nucleotide accessibility scores at the first and second levels of analysis are mean scores. As a result, two nucleotides with identical accessibility scores may have markedly different numbers of unpaired predicted secondary structures at each step of transcriptional analysis. In analyzing accessibility for AON binding, it may be important to take into account steps of transcriptional analysis in which a nucleotide is predicted to have total absence of unpaired secondary structures, i.e. the nucleotide is predicted to be completely inaccessible or “engaged” at the particular step of transcriptional analysis (Figure 2B). For the purpose of analysis, at every step of transcriptional analysis, each nucleotide in the AON target site which is engaged may then be depicted in a plot as illustrated in Figure 3. Table S3 of the Online Supporting Information tabulates these plots for all the AON target sites analyzed.

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.

View Article: PubMed Central - PubMed

Affiliation: Bioinformatics Institute, Singapore, Singapore.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus