Modulation of the RNA Interference Activity Using Central Mismatched siRNAs and Acyclic Threoninol Nucleic Acids (aTNA) Units
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design and Thermodynamic Properties of siRNA Carrying L-Threoninol Monomers
Antisense ..ZW.. | Sense ..XY.. | IC50 (pM) ± SD | Tm (°C) ± SD | ΔTm (wt) | ΔTm (Parent) | |
---|---|---|---|---|---|---|
wt | ..U.. | ..A.. | 9.6 ± 0.5 | 67.8 ± 0.3 | -- | -- |
wtU9 | ..U.. | ..A.. | 15.4 ± 0.7 | 60.4 ± 0.2 | 7.6 | 7.6 |
wtC9 | ..U.. | ..A.. | 30.7 ± 0.6 | 59.5 ± 0.4 | 8.5 | 8.5 |
wtG9 | ..U.. | ..A.. | 15.8 ± 0.2 | 65.9 ± 0.2 | 2.1 | 2.1 |
wtU10 | ..U.. | ..A.. | No active | 58.2 ± 0.4 | 9.6 | 9.6 |
wtC10 | ..U.. | ..A.. | No active | 60.0 ± 0.1 | 7.8 | 7.8 |
wtG10 | ..U.. | ..A.. | 101 ± 0.7 | 64.5 ± 0.6 | 3.5 | 3.5 |
T10A10 | ..U.. | ..A.. | 111 ± 0.8 | 58.3 ± 0.3 | 9.5 | -- |
T11A9 | ..U.. | ..A.. | 20.2 ± 0.6 | 59.1 ± 0.3 | 8.7 | -- |
T10U10 | ..U.. | ..A.. | 216 ± 0.6 | 55.2 ± 0.1 | 12.6 | 3.1 |
T10C10 | ..U.. | ..A.. | 277 ± 0.9 | 54.1 ± 0.4 | 13.7 | 4.2 |
T10G10 | ..U.. | ..A.. | 110 ± 0.9 | 55.7 ± 0.3 | 10.8 | 1.3 |
T11U9 | ..U.. | ..A.. | 35.6 ± 0.4 | 56.4 ± 0.2 | 11.4 | 2.7 |
T11C9 | ..U.. | ..A.. | 57.3 ± 0.5 | 55.6 ± 0.2 | 12.2 | 3.5 |
T11G9 | ..U.. | ..A.. | 26.5 ± 0.8 | 58.4 ± 0.5 | 9.4 | 0.7 |
2.2. Impact of Mismatches and/or L-Threoninol Modifications on the Silencing Activity of siRNAs
2.3. Central Mismatched siRNA: Trying to Bias the Silence
2.4. Central L-Threoninol Modified siRNAs act through an Ago2-Mediated Mechanism
2.5. Single-Stranded siRNAs Experiments
2.6. Silencing Asymmetry and “Strand-Blocking” Effect of TL Modification
Antisense ..A.. | Sense ..K.. | IC50 (pM) ± SD | Tm (°C) ± SD | Δ Tm (wt) | |
---|---|---|---|---|---|
wt | ..A.. | ..U.. | 9.6 ± 0.5 | 67.8 ± 0.3 | -- |
wtT2 | ..A.. | ..TL.. | 13.4 ± 0.3 | 66.7 ± 0.3 | 1.1 |
3. Experimental Section
3.1. RNA Synthesis
3.2. Deprotection and Purification of Unmodified and Modified RNA Oligonucleotide
3.3. SiRNA Preparation
3.4. Thermal Denaturation Studies
3.5. Cells
3.6. PsiCHECK2 on-/off-Target Reporters
3.7. Transfection and Luciferase Assay
3.8. Ago2-Mediated Silencing Assay
3.9. Single-Stranded siRNA 5'-End Phosphorylation
3.10. Isolation of RNA and RT-qPCR
3.11. Statistical Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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- Sample Availability: Samples of the compounds are not available from the authors.
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Alagia, A.; Terrazas, M.; Eritja, R. Modulation of the RNA Interference Activity Using Central Mismatched siRNAs and Acyclic Threoninol Nucleic Acids (aTNA) Units. Molecules 2015, 20, 7602-7619. https://doi.org/10.3390/molecules20057602
Alagia A, Terrazas M, Eritja R. Modulation of the RNA Interference Activity Using Central Mismatched siRNAs and Acyclic Threoninol Nucleic Acids (aTNA) Units. Molecules. 2015; 20(5):7602-7619. https://doi.org/10.3390/molecules20057602
Chicago/Turabian StyleAlagia, Adele, Montserrat Terrazas, and Ramon Eritja. 2015. "Modulation of the RNA Interference Activity Using Central Mismatched siRNAs and Acyclic Threoninol Nucleic Acids (aTNA) Units" Molecules 20, no. 5: 7602-7619. https://doi.org/10.3390/molecules20057602