Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design and Preparation of the Covalent V7t1 Dimers
2.2. Gel Electrophoresis Analysis
2.3. Size exclusion (SE) Chromatography Analysis
2.4. Spectroscopic Characterization and Singular Value Decomposition (SVD) Analyses of Covalent V7t1 Dimers Folding
2.4.1. UV Analysis
2.4.2. CD Spectra and SVD Analysis
2.4.3. CD Thermal Denaturation/Renaturation Measurements
2.5. Binding Experiments with VEGF165 Protein
2.6. In vitro Bioactivity Studies
3. Conclusions
4. Materials and Methods
4.1. General Methods
- ▪
- V7t1 (5′TGTGGGGGTGGACGGGCCGGGTAGA3′);
- ▪
- bisV7t1T7 (5′V7t13′TTTTTTT5′V7t13′);
- ▪
- bisV7t1HEG2 (5′V7t13′C24H51O20P35′V7t13′);
- ▪
- bisV7t1TEG2D (5′V7t13′C25H53N2O24P53′V7t15′).
4.2. Preparation of the Oligonucleotide Samples
4.3. Gel Electrophoresis Analysis
4.4. Size Exclusion Chromatography
4.5. UV Spectroscopy
4.6. Circular Dichroism (CD) Spectroscopy
4.7. Electrophoresis Mobility Shift Assay (EMSA)
4.8. In vitro Biological Evaluation
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Name | Sequence | Linker Length |
---|---|---|
bisV7t1T7 | 42 | |
bisV7t1HEG2 | 40 | |
bisV7t1TEG2D | 40 |
Prediction of G4 Topologies Relative Abundance from SVD Analysis of CD Spectra | |||
---|---|---|---|
HEPES/Na+ | |||
Parallel (%) | Hybrid (%) | Antiparallel (%) | |
N.A. V7t1 | 100 | 1 | 0 |
A. V7t1 | 36 | 0 | 63 |
N.A. bisV7t1T7 | 72 | 7 | 20 |
A. bisV7t1T7 | 32 | 0 | 67 |
N.A. bisV7t1HEG2 | 78 | 0 | 23 |
A. bisV7t1HEG2 | 34 | 0 | 65 |
N.A. bisV7t1TEG2D | 100 | 1 | 0 |
A. bisV7t1TEG2D | 75 | 26 | 0 |
TRIS/K+ | |||
N.A. V7t1 | 100 | 1 | 0 |
A. V7t1 | 92 | 2 | 6 |
N.A. bisV7t1T7 | 97 | 4 | 0 |
A. bisV7t1T7 | 45 | 38 | 16 |
N.A. bisV7t1HEG2 | 100 | 1 | 0 |
A. bisV7t1HEG2 | 100 | 1 | 0 |
N.A. bisV7t1TEG2D | 100 | 1 | 0 |
A. bisV7t1TEG2D | 100 | 1 | 0 |
Molecule | IC50 (µM) |
---|---|
V7t1 | >20 µM |
bisV7t1T7 | 19 µM |
bisV7t1HEG2 | >20 µM |
bisV7t1TEG2D | >20 µM |
control 24-mer | >20 µM |
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Riccardi, C.; Musumeci, D.; Platella, C.; Gaglione, R.; Arciello, A.; Montesarchio, D. Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs. Int. J. Mol. Sci. 2020, 21, 1963. https://doi.org/10.3390/ijms21061963
Riccardi C, Musumeci D, Platella C, Gaglione R, Arciello A, Montesarchio D. Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs. International Journal of Molecular Sciences. 2020; 21(6):1963. https://doi.org/10.3390/ijms21061963
Chicago/Turabian StyleRiccardi, Claudia, Domenica Musumeci, Chiara Platella, Rosa Gaglione, Angela Arciello, and Daniela Montesarchio. 2020. "Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs" International Journal of Molecular Sciences 21, no. 6: 1963. https://doi.org/10.3390/ijms21061963
APA StyleRiccardi, C., Musumeci, D., Platella, C., Gaglione, R., Arciello, A., & Montesarchio, D. (2020). Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs. International Journal of Molecular Sciences, 21(6), 1963. https://doi.org/10.3390/ijms21061963