Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs
The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical mod...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-09-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253122001482 |
| _version_ | 1811242727866630144 |
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| author | Tomasz Czapik Julita Piasecka Ryszard Kierzek Elzbieta Kierzek |
| author_facet | Tomasz Czapik Julita Piasecka Ryszard Kierzek Elzbieta Kierzek |
| author_sort | Tomasz Czapik |
| collection | DOAJ |
| description | The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical modifications aiming to improve the RNA cleavage activity of ribozymes. We introduced an additional hairpin motif and attempted to select ribozyme-target pairs with sequence features that enable the potential formation of the trans-Hoogsteen interactions that are present in full-length, highly active hammerhead ribozymes. We placed structurally defined guanosine analogs into the ribozyme catalytic core. Herein, the significantly improved synthesis of 2′-deoxy-2′-fluoroarabinoguanosine derivatives is described. The most potent hammerhead ribozymes were applied to chimeric short hairpin RNA (shRNA)-ribozyme plasmid constructs to improve the antiviral activity of the two components. The modified hammerhead ribozymes showed moderate cleavage activity. Treatment of IAV-infected Madin-Darby canine kidney (MDCK) cells with the plasmid constructs resulted in significant inhibition of virus replication. Real-time PCR analysis revealed a significant (80%–88%) reduction in viral RNA when plasmids carriers were used. A focus formation assay (FFA) for chimeric plasmids showed inhibition of virus replication by 1.6–1.7 log10 units, whereas the use of plasmids carrying ribozymes or shRNAs alone resulted in lower inhibition. |
| first_indexed | 2024-04-12T13:55:07Z |
| format | Article |
| id | doaj.art-c3b9450fc42640b1a7cb711e7219f250 |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-12T13:55:07Z |
| publishDate | 2022-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-c3b9450fc42640b1a7cb711e7219f2502022-12-22T03:30:24ZengElsevierMolecular Therapy: Nucleic Acids2162-25312022-09-01296474Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifsTomasz Czapik0Julita Piasecka1Ryszard Kierzek2Elzbieta Kierzek3Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PolandInstitute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland; Corresponding author Elzbieta Kierzek, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical modifications aiming to improve the RNA cleavage activity of ribozymes. We introduced an additional hairpin motif and attempted to select ribozyme-target pairs with sequence features that enable the potential formation of the trans-Hoogsteen interactions that are present in full-length, highly active hammerhead ribozymes. We placed structurally defined guanosine analogs into the ribozyme catalytic core. Herein, the significantly improved synthesis of 2′-deoxy-2′-fluoroarabinoguanosine derivatives is described. The most potent hammerhead ribozymes were applied to chimeric short hairpin RNA (shRNA)-ribozyme plasmid constructs to improve the antiviral activity of the two components. The modified hammerhead ribozymes showed moderate cleavage activity. Treatment of IAV-infected Madin-Darby canine kidney (MDCK) cells with the plasmid constructs resulted in significant inhibition of virus replication. Real-time PCR analysis revealed a significant (80%–88%) reduction in viral RNA when plasmids carriers were used. A focus formation assay (FFA) for chimeric plasmids showed inhibition of virus replication by 1.6–1.7 log10 units, whereas the use of plasmids carrying ribozymes or shRNAs alone resulted in lower inhibition.http://www.sciencedirect.com/science/article/pii/S2162253122001482MT: oligonucleotides: therapies and applicationshammerhead ribozymeinfluenza virusRNA secondary structureRNA conserved structural motifsmodified nucleosides |
| spellingShingle | Tomasz Czapik Julita Piasecka Ryszard Kierzek Elzbieta Kierzek Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs Molecular Therapy: Nucleic Acids MT: oligonucleotides: therapies and applications hammerhead ribozyme influenza virus RNA secondary structure RNA conserved structural motifs modified nucleosides |
| title | Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs |
| title_full | Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs |
| title_fullStr | Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs |
| title_full_unstemmed | Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs |
| title_short | Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs |
| title_sort | structural variants and modifications of hammerhead ribozymes targeting influenza a virus conserved structural motifs |
| topic | MT: oligonucleotides: therapies and applications hammerhead ribozyme influenza virus RNA secondary structure RNA conserved structural motifs modified nucleosides |
| url | http://www.sciencedirect.com/science/article/pii/S2162253122001482 |
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