Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro
Abstract
:1. Introduction
2. Results
2.1. Evaluation of α-l-LNA AOs to Induce Exon-23 Skipping
2.2. Evaluation of Cytotoxicity of AOs
3. Discussion
4. Materials and Methods
4.1. Melting Temperature Analysis
4.2. Cell Culture and Transfection
4.3. RNA Extraction and RT-PCR
4.4. Cell Viability and Cytotoxicity Assay
4.5. Evaluation of Efficiency of Exon Skipping
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
AO | Antisense oligonucleotide |
α-l-LNA | Alpha-L-locked nucleic acid |
DMD | Duchenne muscular dystrophy |
References
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AO Name | Sequence (5′-3′ direction) | Tm (°C) |
---|---|---|
NAC 9078 (20mer) | GGC CAA ACC UCG GCαT UAαC CαT | 65.2 |
NAC 9079 (18mer) | GCC AAA CCU CGG αCUαT ACαC | 64.2 |
NAC 9080 (18mer) | GαCC AAA αCCU αCGG CαTU AαCC | 68.8 |
NAC 9081 (16mer) | CCA AAC CUC GGαC UαTA αC | 59.6 |
2′-O-MePS (20mer) | GGCCAAACCUCGGCUUACCU | 60.8 |
2′-O-MePS (18mer) | GCCAAACCUCGGCUUACC | 57.7 |
2′-O-MePS (16mer) | CCAAACCUCGGCUUAC | 53.1 |
AO Name | Percentage of Exon 23 Skipping (%) | Percentage of Dual Exon 22/23 Skipping (%) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
2.5 nM | 5 nM | 12.5 nM | 25 nM | 50 nM | 100 nM | 2.5 nM | 5 nM | 12.5 nM | 25 nM | 50 nM | 100 nM | |
NAC 9078 | 6 (2.93) | 16 (2.23) | 49 (12.41) | 56 (11.68) | 66 (4.21) | 74 (11.92) | 5 (6.95) | 5 (3.36) | 4 (4.51) | 16 (5.33) | 20 (8.13) | 19 (8.50) |
NAC9079 | 13 (13.23) | 12 (7.51) | 44 (15.76) | 24 (26.30) | 74 (15.27) | 71 (10.53) | 2 (2.01) | 4 (2.27) | 12 (13.79) | 10 (9.26) | 13 (10.30) | 14 (12.94) |
NAC 9080 | 15 (17.13) | 23 (15.42) | 24 (18.90) | 55 (15.52) | 68 (11.23) | 66 (17.12) | 4 (3.62) | 4 (2.53) | 13 (8.43) | 11 (8.79) | 21 (5.55) | 29 (18.86) |
NAC 9081 | 5 (4.08) | 6 (4.35) | 12 (8.10) | 19 (19.71) | 40 (9.13) | 60 (9.29) | 4 (5.02) | 4 (5.26) | 6 (7.64) | 5 (4.37) | 15 (2.95) | 22 (8.75) |
2′-O-MePS (20mer) | 7 (3.10) | 16 (15.06) | 26 (20.88) | 46 (22.81) | 44 (29.25) | 60 (17.13) | 5 (7.68) | 1 (0.34) | 1 (0.18) | 7 (5.06) | 22 (6.65) | 30 (16.92) |
2′-O-MePS (18mer) | 10 (7.96) | 10 (6.83) | 18 (17.62) | 48 (16.86) | 64 (15.38) | 69 (12.22) | 6 (7.39) | 5 (5.99) | 11 (2.41) | 12 (9.07) | 19 (13.45) | 20 (11.89) |
2′-O-MePS (16mer) | 4 (1.79) | 4 (1.04) | 11 (4.32) | 28 (27.75) | 35 (29.98) | 50 (21.53) | 8 (11.34) | 4 (5.10) | 9 (5.06) | 23 (12.06) | 19 (14.58) | 20 (14.81) |
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Raguraman, P.; Wang, T.; Ma, L.; Jørgensen, P.T.; Wengel, J.; Veedu, R.N. Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro. Int. J. Mol. Sci. 2020, 21, 2434. https://doi.org/10.3390/ijms21072434
Raguraman P, Wang T, Ma L, Jørgensen PT, Wengel J, Veedu RN. Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro. International Journal of Molecular Sciences. 2020; 21(7):2434. https://doi.org/10.3390/ijms21072434
Chicago/Turabian StyleRaguraman, Prithi, Tao Wang, Lixia Ma, Per Trolle Jørgensen, Jesper Wengel, and Rakesh N. Veedu. 2020. "Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro" International Journal of Molecular Sciences 21, no. 7: 2434. https://doi.org/10.3390/ijms21072434