Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs

Molecular Therapy: Nucleic Acids

Tomasz Czapik¹  Ryszard Kierzek¹  Julita Piasecka¹  Elzbieta Kierzek¹ 

[1] Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland;

关键词: MT: oligonucleotides: therapies and applications;    hammerhead ribozyme;    influenza virus;    RNA secondary structure;    RNA conserved structural motifs;    modified nucleosides;   

DOI  :  

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown