Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides
Abstract
:1. Introduction
- The pre-mRNA sequence is interrogated by one or more in silico prediction programs to identify potential splice enhancer or silencer motifs.
- Antisense oligonucleotides, typically 20 to 25 mers, are designed to anneal to the target motifs and synthesised as 2′-O-methyl (2-OMe) modified bases on a phosphorothioate (PS) backbone.
- The test compounds are complexed with cationic liposome preparations and transfected into cells.
- After incubation, total RNA is extracted and the target transcript is amplified using RT-PCR to assess differences in pre-mRNA processing, with and without AO treatment.
- Oligomers shown to induce the desired changes in pre-mRNA processing are further refined by micro-walking around the annealing site and/or altering AO length.
- Transfection studies over a range of concentrations are performed to identify compound(s) that modify splicing in a dose-dependent manner, and at the lowest concentration.
2. Results
Guidelines for Developing Splice Switching AOs
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Antisense Oligonucleotides (AOs)
4.3. 2′-O-Methyl Phosphorothioate AO Transfection
4.4. RT-PCR
Author Contributions
Funding
Conflicts of Interest
References
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Cell Lines | Transfection Reagents |
---|---|
Dermal fibroblasts | Lipofectin™, Lipofectamine™ 3000 |
Myoblasts and myotubes | Lipofectamine™ 2000 |
Lymphoblasts and lymphocytes | Nucleofection P3 Primary Cell Kit |
Huh7 | Lipofectamine™ 3000, Lipofectamine™ RNAiMax |
HEK293 | Lipofectamine™ 3000 |
H2k mdx | Lipofectin™ |
MO3.13 | Lipofectamine™ 3000 |
iPSCs and neural stem cells | Lipofectamine™ Stem |
Name | Sequence (5′ – 3′) |
---|---|
ITGA4 H3A(+30+49) | UCUCUCUCUUCCAAACAAGU |
ITGA4 H3A(-18+7) | GGGCUACCUAUAGCAUGUGAAAAUA |
ITGA4 H3A(+20+39) | CCAAACAAGUCUUUCCACAA |
ITGA4 H3A(+46+70) | GUGACCCCCAACCACUGAUUGUCUC |
ITGA4 H3A(+41+65) | CCCCAACCACUGAUUGUCUCUCUCU |
ITGA4 H3A(+51+75) | AAAGUGUGACCCCCAACCACUGAUU |
ITGA4 H3D(+6-19) | GACCAGUUCCAAUACCUACCACGAU |
ITGA4 H3D(+11-14) | GUUCCAAUACCUACCACGAUGGAUC |
ITGA4 H3D(+1-24) | CUGUGGACCAGUUCCAAUACCUACC |
Ctl | GGAUGUCCUGAGUCUAGACCCUCCG |
Name | Sequence (5′ – 3′) | Amplification Performed after Treatment with the Following AOs |
---|---|---|
ITGA4 ex1_F | gagagcgcgctgctttaccagg | All AOs |
ITGA4 ex10_R | gccatcattgtcaatgtcgcca | |
ITGA4 ex1_F | gagagcgcgctgctttaccagg | ITGA4 H3A(+30+49) |
ITGA4 ex4_R | ggcactccatagcaaccacc |
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Aung-Htut, M.T.; McIntosh, C.S.; Ham, K.A.; Pitout, I.L.; Flynn, L.L.; Greer, K.; Fletcher, S.; Wilton, S.D. Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides. Int. J. Mol. Sci. 2019, 20, 5030. https://doi.org/10.3390/ijms20205030
Aung-Htut MT, McIntosh CS, Ham KA, Pitout IL, Flynn LL, Greer K, Fletcher S, Wilton SD. Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides. International Journal of Molecular Sciences. 2019; 20(20):5030. https://doi.org/10.3390/ijms20205030
Chicago/Turabian StyleAung-Htut, May T., Craig S. McIntosh, Kristin A. Ham, Ianthe L. Pitout, Loren L. Flynn, Kane Greer, Sue Fletcher, and Steve D. Wilton. 2019. "Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides" International Journal of Molecular Sciences 20, no. 20: 5030. https://doi.org/10.3390/ijms20205030