RNA interference-mediated simultaneous silencing of four genes using cross-shaped RNA.

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.

Branched, tripartite-interfering RNAs silence multiple target genes with long guide strands.

Structural modifications could provide classical small interfering RNA (siRNA) structure with several advantages, including reduced off-target effects and increased silencing activity. Thus, RNA interference (RNAi)-triggering molecules with diverse structural modifications have been investigated by introducing variations on duplex length and overhang structure. However, most of siRNA structural variants are based on the linear duplex structure. In this study, we introduce a branched, non-linear tripartite-interfering RNA (tiRNA) structure that could induce silencing of multiple target genes. Surprisingly, the gene silencing by tiRNA structure does not require Dicer-mediated processing into smaller RNA units, and the 38-nt-long guide strands can trigger specific gene silencing through the RNAi machinery in mammalian cells. tiRNA also shows improved gene silencing potency over the classical siRNA structure when complexed with cationic delivery vehicles due to the enhanced intracellular delivery. These results demonstrate that tiRNA is a novel RNA nanostructure for executing multi-target gene silencing with increased potency, which could be utilized as a structural platform to develop efficient anticancer or antiviral RNAi therapeutics.

Enhanced intracellular delivery and multi-target gene silencing triggered by tripodal RNA structures.

BACKGROUND: The development of gene interfering RNA (iRNA) molecules such as small interfering RNAs (siRNAs) and antagomirs provides promising therapeutic modalities for targeting specific mRNAs and microRNAs (miRNAs) involved in disease mechanisms. Therapeutic iRNA strategy against cancer or hypermutable viruses prefers targeting multiple genes simultaneously to achieve synergistic inhibition and to prevent resistance. METHODS: In the present study, we report chemically synthesized, multi-target gene interfering RNA structures based upon branched, tripodal interfering RNAs (termed T-tiRNAs). RESULTS: The T-tiRNAs could simultaneously inhibit up to three different mRNAs or miRNAs by harboring three siRNA or antagomir units. Moreover, when complexed with cationic delivery vehicles, T-tiRNAs showed enhanced gene interfering activity over conventional siRNAs or antagomirs as a result of increased intracellular delivery. CONCLUSIONS: The data obtained in the present study provide an example of synthetic multi-functional RNA structures that enable multiple gene interference in mammalian cells, which could become powerful tools for an efficient combinatorial iRNA strategy.

Structural diversity repertoire of gene silencing small interfering RNAs.

Since the discovery of double-stranded (ds) RNA-mediated RNA interference (RNAi) phenomenon in Caenorhabditis elegans, specific gene silencing based upon RNAi mechanism has become a novel biomedical tool that has extended our understanding of cell biology and opened the door to an innovative class of therapeutic agents. To silence genes in mammalian cells, short dsRNA referred to as small interfering RNA (siRNA) is used as an RNAi trigger to avoid nonspecific interferon responses induced by long dsRNAs. An early structure-activity relationship study performed in Drosophila melanogaster embryonic extract suggested the existence of strict siRNA structural design rules to achieve optimal gene silencing. These rules include the presence of a 3' overhang, a fixed duplex length, and structural symmetry, which defined the structure of a classical siRNA. However, several recent studies performed in mammalian cells have hinted that the gene silencing siRNA structure could be much more flexible than that originally proposed. Moreover, many of the nonclassical siRNA structural variants reported improved features over the classical siRNAs, including increased potency, reduced nonspecific responses, and enhanced cellular delivery. In this review, we summarize the recent progress in the development of gene silencing siRNA structural variants and discuss these in light of the flexibility of the RNAi machinery in mammalian cells.

Long double-stranded RNA-mediated RNA interference and immunostimulation: long interfering double-stranded RNA as a potent anticancer therapeutics.

In most applications, small interfering RNAs are designed to execute specific gene silencing via RNA interference (RNAi) without triggering nonspecific responses such as immunostimulation. However, in anticancer therapeutics, immunostimulation combined with specific oncogene silencing could be beneficial, resulting in the synergistic inhibition of cancer cell growth. In this study, we report an immunostimulatory long double-stranded RNA (dsRNA) structure with the ability to trigger RNAi-mediated specific target gene silencing, termed as long interfering dsRNA (liRNA). liRNA targeting Survivin mRNA not only efficiently and specifically triggered target gene silencing via RNAi, but also stimulated the protein kinase R pathway to induce the expression of interferon ß. As a result, the ability of Survivin-targeting liRNA to inhibit cancer cell growth was superior over conventional small interfering RNA or nontargeting dsRNA structures. Our results thus provide a simple yet efficient dual function immunostimulatory RNAi-triggering structure, which is potentially applicable for the development of anticancer therapeutics.

Dual-target gene silencing by using long, synthetic siRNA duplexes without triggering antiviral responses.

Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of nonspecific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without nonspecific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.

Asymmetric shorter-duplex siRNA structures trigger efficient gene silencing with reduced nonspecific effects.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that mediate efficient gene silencing in a sequence-specific manner by utilizing the endogenous RNA interference (RNAi) pathway. The current standard synthetic siRNA structure harbors a 19-base-pair duplex region with 3' overhangs of 2 nucleotides (the so-called 19+2 form). However, the synthetic 19+2 siRNA structure exhibits several sequence-independent, nonspecific effects, which has posed challenges to the development of RNAi therapeutics and specific silencing of genes in research. In this study, we report on the identification of truncated siRNA backbone structures with duplex regions shorter than 19 bp (referred to as asymmetric shorter-duplex siRNAs or asiRNAs) that can efficiently trigger gene silencing in human cell lines. Importantly, this asiRNA structure significantly reduces nonspecific effects triggered by conventional 19+2 siRNA scaffold, such as sense-strand-mediated off-target gene silencing and saturation of the cellular RNAi machinery. Our results suggest that this asiRNA structure is an important alternative to conventional siRNAs for both functional genomics studies and therapeutic applications.

A structure-activity relationship study of siRNAs with structural variations.

Specific knock-down of cellular gene expression using small interfering RNAs (siRNAs) is a powerful gene silencing technique in mammalian cells. Early siRNAs were double stranded, and 19-21bp in length, but several variations in siRNA structure have been introduced to achieve better silencing efficiency. In addition, siRNA modules have been incorporated into higher-order RNA structures to generate multi-functional RNA molecules. The effects of such structural variations on the activities of siRNAs have not been thoroughly studied. Here, we present a structure-activity relationship study of siRNA structural variants. Specifically, we focus on the effect on silencing efficiency of the attachment of extra, target-unrelated sequences to the conventional short duplex siRNA structure. Interestingly, while some siRNA structural variants efficiently silence target gene expression, others show a reduction in or a complete lack of silencing activity. Off-target effects and innate immune responses triggered by siRNA structural variants were also measured. In vitro Dicer cleavage reactions show that all siRNA structural variants are substrates of Dicer, but digestion patterns vary. To our knowledge, this is the first systematic structure-activity relationship analysis of siRNAs bearing structural variations. Our results provide useful guidelines for the design of siRNA structural variants and for the construction of complex RNA molecules bearing functional siRNA modules.