Optimization of Bifunctional Antisense Oligonucleotides for Regulation of Mutually Exclusive Alternative Splicing of PKM Gene
Abstract
:1. Introduction
2. Results and Discussion
2.1. Oligonucleotides Screening and Binding Affinity Determination
2.2. Cell Line Results
2.2.1. 5′ UAGGU 3′ Regulatory Sequence
2.2.2. 5′ CAGGUAAGU 3′ Regulatory Sequence
2.2.3. Branched BASOs
3. Material and Methods
3.1. Oligonucleotide Synthesis
3.2. hnRNP A1 Protein Production
3.3. Electrophoretic Mobility Shift Assay (EMSA)—Oligonucleotides Screening
3.4. Electrophoretic Mobility Shift Assay (EMSA)-Kd Determination
3.5. UV Melting Experiments
3.6. Cell Culture and Oligonucleotides Transfection
3.7. RT-qPCR Analysis
3.8. qPCR Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Name | Sequence (5′ -> 3′) |
---|---|
A2 | CAGGUAAGU CAGGUAAGU |
A3 | CAGGUAAGU CAGGUAAGU CAGGUAAGU |
B2 | CAGGUGAGU CAGGUGAGU |
B3 | CAGGUGAGU CAGGUGAGU CAGGUGAGU |
C2 | UAGGA UAGGA |
C4 | UAGGA UAGGA UAGGA UAGGA |
D2 | UAGGU UAGGU |
D4 | UAGGU UAGGU UAGGU UAGGU |
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Bartyś, N.; Pasternak, A.; Lisowiec-Wąchnicka, J. Optimization of Bifunctional Antisense Oligonucleotides for Regulation of Mutually Exclusive Alternative Splicing of PKM Gene. Molecules 2022, 27, 5682. https://doi.org/10.3390/molecules27175682
Bartyś N, Pasternak A, Lisowiec-Wąchnicka J. Optimization of Bifunctional Antisense Oligonucleotides for Regulation of Mutually Exclusive Alternative Splicing of PKM Gene. Molecules. 2022; 27(17):5682. https://doi.org/10.3390/molecules27175682
Chicago/Turabian StyleBartyś, Natalia, Anna Pasternak, and Jolanta Lisowiec-Wąchnicka. 2022. "Optimization of Bifunctional Antisense Oligonucleotides for Regulation of Mutually Exclusive Alternative Splicing of PKM Gene" Molecules 27, no. 17: 5682. https://doi.org/10.3390/molecules27175682