Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance
Abstract
:1. Introduction
2. Results
2.1. Design Strategy of Dual Conjugates
2.2. Hybridization of Dual Conjugates with miRNA Targets
2.3. Cleavage Profile and Kinetics of miRNA Cleavage by Dual Conjugates
2.4. The Effect of Mg2+ and K+ Concentration on the Ribonuclease Activity of Dual Conjugates
2.5. The Effect of pH on the Ribonuclease Activity of Dual Conjugates
2.6. Efficiency and Specificity of miRNA Cleavage by Dual Conjugates in the Presence of RNase H
3. Discussion
4. Materials and Methods
4.1. Oligonucleotides
4.2. Synthesis of the Conjugates
4.3. RNA Labelling
4.4. Gel-Retardation assay
4.5. Ribonuclease Activity Assay
4.6. Ribonuclease Activity Assay Using Dual Conjugates and RNase H
4.7. Ribonuclease Activity Assay at Different Concentrations of MgCl2
4.8. Ribonuclease Activity Assay at Different Concentrations of KCl
4.9. pH Profile of Ribonuclease Activity of DCs
4.10. Stability of DCs and ONs in 10% FBS
4.11. Statistics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the compounds are available from the authors. |
Conjugate | Sequence 5′→3′ |
---|---|
DC | |
miRNA-21-DC | TCAACATC–linker 1–[GRLRL]2–linker 2–GATAAGCTA |
miRNA-18a-DC | TATCTGC–linker 1–[GRLRL]2–linker 2–ATGCACCTTA |
miRNA-17-DC | CTACCTGC–linker 1–[GRLRL]2–linker 2–AAGCACTTTG |
miRNA-155-DC | ACCCCTAT–linker 1–[GRLRL]2–linker 2–TTAGCATTAA |
SC | |
miRNA-21-SC | [GRLRL]2–linker 2–GATAAGCTA |
miRNA-18a-SC | [GRLRL]2–linker 2–ATGCACCTTA |
miRNA-17-SC | [GRLRL]2–linker 2–AAGCACTTTG |
miRNA-155-SC | [GRLRL]2–linker 2–TTAGCATTAA |
ON | |
miRNA-21-ON | TCAACATC–TEG–TEG–GATAAGCTA |
miRNA-18a-ON | TATCTGC–TEG–TEG–ATGCACCTTA |
miRNA-17-ON | CTACCTGC–TEG–TEG–AAGCACTTTG |
miRNA-155-ON | ACCCCTAT–TEG–TEG–TTAGCATTAA |
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Patutina, O.; Chiglintseva, D.; Bichenkova, E.; Gaponova, S.; Mironova, N.; Vlassov, V.; Zenkova, M. Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance. Molecules 2020, 25, 2459. https://doi.org/10.3390/molecules25102459
Patutina O, Chiglintseva D, Bichenkova E, Gaponova S, Mironova N, Vlassov V, Zenkova M. Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance. Molecules. 2020; 25(10):2459. https://doi.org/10.3390/molecules25102459
Chicago/Turabian StylePatutina, Olga, Daria Chiglintseva, Elena Bichenkova, Svetlana Gaponova, Nadezhda Mironova, Valentin Vlassov, and Marina Zenkova. 2020. "Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance" Molecules 25, no. 10: 2459. https://doi.org/10.3390/molecules25102459