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

Abstract

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.

Keywords: MT: oligonucleotides: therapies and applications; RNA conserved structural motifs; RNA secondary structure; fluoroarabinonucleosides; hammerhead ribozyme; influenza virus; modified nucleosides; shRNA.

Graphical abstract

Figure 1

Hammerhead ribozyme type II scheme (A) Model hammerhead ribozyme secondary structure. (B) Possible tertiary interactions between loop L2 and stem I are enabled in the Y conformation. The target sequence is marked in green, with the black arrow indicating the cleavage site. Two nucleosides, 2.2 and 2.3, of tetraloop L2 are marked in yellow. Only the sequence of the conserved catalytic core and target NUH are shown.

Figure 2

Model hammerhead ribozymes with the RNA target sequences Target RNA sequences are shown in green, and the side arms of the ribozymes are shown in orange. Nucleosides that may be involved in trans-Hoogsteen iterations are marked in red, and changes in hammerhead stem II and loop L2 are marked in yellow.

Figure 3

Antiviral activity of the ribozymes in MDCK cell culture based on real-time PCR quantitative analysis of viral RNA (A) Sequential and structural variants of the ribozymes targeting (+)RNA5 region 184–200. (B) Chemically modified variants of the ribozyme targeting (+)RNA5 region 184–200 are shown. (C) Ribozymes targeting (+)RNA5 region 615–704 are shown. The viral RNA level in the ribozyme-treated samples was compared with the Lipofectamine-treated control (LF). INF denotes the untreated infected cells. Error bars represent the standard error of the mean (SEM). An unpaired two-tailed t test was performed for statistical comparisons (∗p < 0.05 and ∗∗p < 0.01).

Figure 4

Antiviral potential of plasmid constructs (A and B) Inhibitory potential of the tested plasmid constructs based on quantitative real-time PCR analysis of viral RNA in samples treated with (A) single-molecule constructs and (B) shRNA-ribozyme constructs. (C and D) Analysis of infectious viral particles by FFA in samples treated with (C) single-molecule constructs and (D) shRNA-ribozyme constructs. Error bars represent the SEM. An unpaired two-tailed t test was performed for statistical comparisons (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).