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Optimizing splice-switching oligomer sequences using 2'-O-methyl phosphorothioate chemistry.
Methods Mol Biol. 2012; 867:169-88.MM

Abstract

We have taken an empirical approach in designing splice-switching oligomers to induce targeted dystrophin exon skipping. The nucleotide sequence of the exon under examination is first analyzed for potential exon recognition motifs and then a set of oligomers complementary to the acceptor and donor splice sites, as well as intra-exonic regions predicted to contain exon splice enhancers, are designed and synthesized as 2'-O-methyl-modified bases on a phosphorothioate backbone (2OMeAOs). The 2OMeAOs can be readily transfected into cultured normal myogenic cells as cationic lipoplexes, and are incubated for 24 h before total RNA extraction and subsequent analysis by semi-quantitative RT-PCR. The amplification conditions used for each dystrophin transcript region under investigation minimize preferential production of shorter amplicons and do not exaggerate the level of induced RT-PCR products, compared to the endogenous dystrophin transcript product. It is imperative that the test oligomers are transfected over a range of concentrations and that the target exon is excised in a reproducible and dose-dependent manner.Once it has been demonstrated that an oligomer can induce some degree of exon skipping, that target region of the pre-mRNA is assumed to be involved in splicing of the exon. A series of overlapping oligomers are prepared and evaluated by transfection into normal myogenic cells at lower concentrations to identify the more effective compounds. Clinical application requires antisense compounds that efficiently modulate splicing at low dosages, delivering the greatest benefits in terms of efficacy, safety, and cost.

Authors+Show Affiliations

Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Perth, Australia.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22454061

Citation

Adkin, Carl, et al. "Optimizing Splice-switching Oligomer Sequences Using 2'-O-methyl Phosphorothioate Chemistry." Methods in Molecular Biology (Clifton, N.J.), vol. 867, 2012, pp. 169-88.
Adkin C, Fletcher S, Wilton SD. Optimizing splice-switching oligomer sequences using 2'-O-methyl phosphorothioate chemistry. Methods Mol Biol. 2012;867:169-88.
Adkin, C., Fletcher, S., & Wilton, S. D. (2012). Optimizing splice-switching oligomer sequences using 2'-O-methyl phosphorothioate chemistry. Methods in Molecular Biology (Clifton, N.J.), 867, 169-88. https://doi.org/10.1007/978-1-61779-767-5_11
Adkin C, Fletcher S, Wilton SD. Optimizing Splice-switching Oligomer Sequences Using 2'-O-methyl Phosphorothioate Chemistry. Methods Mol Biol. 2012;867:169-88. PubMed PMID: 22454061.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Optimizing splice-switching oligomer sequences using 2'-O-methyl phosphorothioate chemistry. AU - Adkin,Carl, AU - Fletcher,Sue, AU - Wilton,Steve D, PY - 2012/3/29/entrez PY - 2012/3/29/pubmed PY - 2012/7/24/medline SP - 169 EP - 88 JF - Methods in molecular biology (Clifton, N.J.) JO - Methods Mol Biol VL - 867 N2 - We have taken an empirical approach in designing splice-switching oligomers to induce targeted dystrophin exon skipping. The nucleotide sequence of the exon under examination is first analyzed for potential exon recognition motifs and then a set of oligomers complementary to the acceptor and donor splice sites, as well as intra-exonic regions predicted to contain exon splice enhancers, are designed and synthesized as 2'-O-methyl-modified bases on a phosphorothioate backbone (2OMeAOs). The 2OMeAOs can be readily transfected into cultured normal myogenic cells as cationic lipoplexes, and are incubated for 24 h before total RNA extraction and subsequent analysis by semi-quantitative RT-PCR. The amplification conditions used for each dystrophin transcript region under investigation minimize preferential production of shorter amplicons and do not exaggerate the level of induced RT-PCR products, compared to the endogenous dystrophin transcript product. It is imperative that the test oligomers are transfected over a range of concentrations and that the target exon is excised in a reproducible and dose-dependent manner.Once it has been demonstrated that an oligomer can induce some degree of exon skipping, that target region of the pre-mRNA is assumed to be involved in splicing of the exon. A series of overlapping oligomers are prepared and evaluated by transfection into normal myogenic cells at lower concentrations to identify the more effective compounds. Clinical application requires antisense compounds that efficiently modulate splicing at low dosages, delivering the greatest benefits in terms of efficacy, safety, and cost. SN - 1940-6029 UR - https://www.unboundmedicine.com/medline/citation/22454061/Optimizing_splice_switching_oligomer_sequences_using_2'_O_methyl_phosphorothioate_chemistry_ L2 - https://dx.doi.org/10.1007/978-1-61779-767-5_11 DB - PRIME DP - Unbound Medicine ER -