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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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Related in: MedlinePlus

Definition of an engaged nucleotide in a particular step of transcriptional analysis.(A) to (C). This is to illustrate that multiple secondary structures of the targeted exon (drawn in green) are predicted in each step of transcriptional analysis, with some of the possible structural motifs shown here. For illustration purpose, a particular nucleotide (marked in red) within an AON target site (green line) is tracked. When this nucleotide is paired (denoted with *), it is not accessible for AON binding. If this nucleotide is paired in all predicted secondary structures, this nucleotide is defined as an engaged nucleotide at this particular step of transcriptional analysis (B).
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pone-0001844-g002: Definition of an engaged nucleotide in a particular step of transcriptional analysis.(A) to (C). This is to illustrate that multiple secondary structures of the targeted exon (drawn in green) are predicted in each step of transcriptional analysis, with some of the possible structural motifs shown here. For illustration purpose, a particular nucleotide (marked in red) within an AON target site (green line) is tracked. When this nucleotide is paired (denoted with *), it is not accessible for AON binding. If this nucleotide is paired in all predicted secondary structures, this nucleotide is defined as an engaged nucleotide at this particular step of transcriptional analysis (B).

Mentions: At first level analysis, each nucleotide within the AON target site, a nucleotide accessibility score will be determined by the following ratio:Note that multiple secondary structures will be predicted in each step of transcriptional analysis, see Figure 2. Hence, for each nucleotide, all secondary structures predicted at every step of transcriptional analysis are included. The accessibility score for the AON target site (L1) will be:The L1 scores for the target sites of the analyzed AONs are tabulated in Table S1 of the Online Supporting Information. The K-S tests failed to show any statistical difference between L1 scores for the target sites of Set A AONs of different grades (Table 1A), which agrees with the results reported in refs. [10] and [61]. On the other hand, the L1 scores for target sites of Set B (++) and (+1) AONs were statistically higher that the L1 scores for target sites of (−) AONs (Table 1B). This result indicates that (++) and (+1) AON target sites are more accessible than (−) AON target sites, and therefore, the L1 score is able to correlate with AON efficacy for Set B AONs. However, as this is not applicable to Set A AONs, we proceeded to the next level of analysis.


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)

Definition of an engaged nucleotide in a particular step of transcriptional analysis.(A) to (C). This is to illustrate that multiple secondary structures of the targeted exon (drawn in green) are predicted in each step of transcriptional analysis, with some of the possible structural motifs shown here. For illustration purpose, a particular nucleotide (marked in red) within an AON target site (green line) is tracked. When this nucleotide is paired (denoted with *), it is not accessible for AON binding. If this nucleotide is paired in all predicted secondary structures, this nucleotide is defined as an engaged nucleotide at this particular step of transcriptional analysis (B).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001844-g002: Definition of an engaged nucleotide in a particular step of transcriptional analysis.(A) to (C). This is to illustrate that multiple secondary structures of the targeted exon (drawn in green) are predicted in each step of transcriptional analysis, with some of the possible structural motifs shown here. For illustration purpose, a particular nucleotide (marked in red) within an AON target site (green line) is tracked. When this nucleotide is paired (denoted with *), it is not accessible for AON binding. If this nucleotide is paired in all predicted secondary structures, this nucleotide is defined as an engaged nucleotide at this particular step of transcriptional analysis (B).
Mentions: At first level analysis, each nucleotide within the AON target site, a nucleotide accessibility score will be determined by the following ratio:Note that multiple secondary structures will be predicted in each step of transcriptional analysis, see Figure 2. Hence, for each nucleotide, all secondary structures predicted at every step of transcriptional analysis are included. The accessibility score for the AON target site (L1) will be:The L1 scores for the target sites of the analyzed AONs are tabulated in Table S1 of the Online Supporting Information. The K-S tests failed to show any statistical difference between L1 scores for the target sites of Set A AONs of different grades (Table 1A), which agrees with the results reported in refs. [10] and [61]. On the other hand, the L1 scores for target sites of Set B (++) and (+1) AONs were statistically higher that the L1 scores for target sites of (−) AONs (Table 1B). This result indicates that (++) and (+1) AON target sites are more accessible than (−) AON target sites, and therefore, the L1 score is able to correlate with AON efficacy for Set B AONs. However, as this is not applicable to Set A AONs, we proceeded to the next level of analysis.

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