RESUMO
This study describes a strategy to develop LNA-modified small interfering RNA (siRNAs) against the highly structured 5' UTR of coxsackievirus B3 (CVB-3), which is an attractive target site due to its high degree of conservation. Accessible sites were identified based on structural models and RNase H assays with DNA oligonucleotides. Subsequently, LNA gapmers, siRNAs, siLNAs and small internally segmented interfering RNA (sisiLNAs) were designed against sites, which were found to be accessible in the in vitro assays, and tested in reporter assays and experiments with the infectious virus. The best siLNA improved viability of infected cells by 92% and exerted good antiviral activity in plaque reduction assays.
Assuntos
Regiões 5' não Traduzidas/antagonistas & inibidores , Antivirais/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Oligonucleotídeos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Regiões 5' não Traduzidas/genética , Animais , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Enterovirus Humano B/genética , Enterovirus Humano B/fisiologia , Células HeLa , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19-27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10-12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo.
