Abstract
Short hairpin RNAs (shRNAs) can induce gene silencing via the RNA interference (RNAi) mechanism. We designed an alternative shRNA molecule with a relatively short base-paired stem that bypasses Dicer and instead is processed by the Argonaute 2 (Ago2) protein into a single guide RNA strand that effectively induces RNAi. We called these molecules AgoshRNAs. Active anti-HIV AgoshRNAs were developed, but their RNAi activity was generally reduced compared with the matching shRNAs. In an attempt to further optimize the AgoshRNA design, we inserted several self-cleaving ribozymes at the 3′ terminus of the transcribed AgoshRNA and evaluated the impact on AgoshRNA processing and activity. The hepatitis delta virus (HDV) ribozyme is efficiently removed from the transcribed AgoshRNAs and generates a uniform 3′ overhang, which translates into the enhanced antiviral activity of these molecules.
Highlights
Since the discovery of the RNA interference (RNAi) process in Caenorhabditis elegans in 1998, RNAi has become an important tool for selective silencing of the expression of target genes in a wide range of mammalian cells.[1,2,3] RNAi can be induced by small interfering RNAs that target complementary mRNAs for degradation or by plasmid-based vectors that express short hairpin RNAs that are processed intracellularly into siRNAs.[4,5,6,7] siRNAs are small RNA duplexes of approximately 21 nucelotides long with a 2 nt overhang at the 30 end
This set includes three AgoshRNAs against overlapping Gag sequences starting at position 1364 (Figure 1B; AgoshGag4-6)
We successfully designed 21 AgoshRNAs that were active in reporter silencing, but only two of these exhibited profound HIV inhibition in spreading virus infections in a T cell line
Summary
Since the discovery of the RNA interference (RNAi) process in Caenorhabditis elegans in 1998, RNAi has become an important tool for selective silencing of the expression of target genes in a wide range of mammalian cells.[1,2,3] RNAi can be induced by small interfering RNAs (siRNAs) that target complementary mRNAs for degradation or by plasmid-based vectors that express short hairpin RNAs (shRNAs) that are processed intracellularly into siRNAs.[4,5,6,7] siRNAs are small RNA duplexes of approximately 21 nucelotides (nt) long with a 2 nt overhang at the 30 end. Man-made shRNAs with a stem of 20–29 base pairs (bp) and a loop of at least 5 nt are transcribed in the nucleus, transported to the cytoplasm by Exportin-5 and processed by Dicer into the active siRNA duplex. The siRNA duplex binds to Argonaute 2 (Ago2) and forms the RNA-induced silencing complex (RISC). The guide strand and the passenger strand are subsequently unwound, and the guide strand is exclusively retained, whereas the passenger strand is degraded or removed from the RISC. Which strand is incorporated as the guide into the RISC is mainly determined by thermodynamic properties of the duplex that are probed by Dicer.[8,9,10] The guide strand, designed to be perfectly complementarity to the target mRNA, will subsequently induce mRNA degradation.[11,12,13]
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