Abstract
Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Herein, the thermodynamics of RISC cofactors and targeting magnesium-rich RNA secondary structures provide additional guidelines for improving siRNA design.
Highlights
Small interfering RNAs that silence genes of infectious diseases are potentially potent drugs
Central to the RNA-induced silencing complex (RISC) catalytic cycle is small interfering RNA (siRNA) binding to the nuclease argonaute 2 (Ago2)
The α-carbon backbone of the post-RISC PAZdomain has a root mean square deviation (RMSD) of 1.81 ± 0.9 Å during the simulations, which is greater than the pre-RISC (RMSD: 0.8 ± 0.3 Å) (Fig. 2A,B)
Summary
Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. The gRNA 3′-terminus binds to the flexible PAZ domain that ejects the pRNA altogether from the RISC At this stage, the solvent exposed positioning of gRNA nt2-nt[4] of the seed region (nt2-nt8) is critical for initial complementary mRNA base pairing[15]. The second and third hypotheses are linked, demonstrating that (ii) effective gRNA-mRNA interactions are initiated and consolidated by magnesium-rich mRNA secondary structures[18] These mRNA secondary structures enable (iii) the RISC catalytic cycle as the primary source for importing magnesium-α and magnesium-γ. Several computational and molecular analyses were performed to investigate these three hypotheses and their potential contributions to siRNA design
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