RNA interference with 2′,4′-bridged nucleic acid analogues

In this study, a number of 2′,4′-BNA- and 2′,4′-BNA^NC-modified siRNAs were designed and synthesized. Their thermal stability, nuclease resistance and gene silencing properties against cultured mammalian cells were evaluated and compared with those of natural siRNAs. The 2′,4′-BNA- and 2′,4′-BNA^NC-modified siRNAs (named siBNA and siBNA^NC, respectively) showed very high T_m values, were remarkably stable in serum sample and showed promising RNAi properties equal to those exhibited by natural siRNAs. Thermally stable siBNAs composed of slightly modified sense and antisense strands were capable of suppressing gene expression equal to that of natural siRNA. A number of modifications on the sense strand by 2′,4′-BNA or 2′,4′-BNA^NC, either consecutively or separated by natural RNA nucleotides, is tolerable in RNAi machinery. Modifications at the Argonauate (Ago2) cleavage site of the sense strand (9–11th positions from the 5′-end of the sense strand) produced variable results depending on siRNA composition. Mostly, modification at the 10th position diminished siRNA activity. In moderately modified siRNAs, modification at the 11th position displayed usual RNAi activity, while modification at the 9th position showed variable results depending on siRNA composition.     

Effective gene silencing activity of prodrug-type 2′-O-methyldithiomethyl siRNA compared with non-prodrug-type 2′-O-methyl siRNA

Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2′-O-modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2′-O-modified siRNA activities were often decreased by modification, since the bulky 2′-O-modifications inhibit to form a RNA-induced silencing complex (RISC). We developed novel prodrug-type 2′-O-methyldithiomethyl (MDTM) siRNA, which is converted into natural siRNA in an intracellular reducing environment. Prodrug-type 2′-O-MDTM siRNAs modified at the 5′-end side including 5′-end nucleotide and the seed region of the antisense strand exhibited much stronger gene silencing effect than non-prodrug-type 2′-O-methyl (2′-O-Me) siRNAs. Furthermore, the resistances for nuclease digestion of siRNAs were actually enhanced by 2′-O-MDTM modifications. Our results indicate that 2′-O-MDTM modifications improve the stability of siRNA in serum and they are able to be introduced at any positions of siRNA.     

Introduction of 2-O-benzyl abasic nucleosides to the 3′-overhang regions of siRNAs greatly improves nuclease resistance

Chemically modified siRNAs containing 2-O-benzyl-1-deoxy-d-ribofuranose (R^H_OBn) in their 3′-overhang region were significantly more resistant towards serum nucleases than siRNAs possessing the natural nucleoside in this region. The knockdown efficacies and binding affinities of these modified siRNAs to the recombinant human Argonaute protein 2 (hAgo2) PAZ domain were comparable with that of siRNA with a thymidine dimer at the 3′-end.     

Synthesis and characterization of small interfering RNAs with haloalkyl groups at their 3′-dangling ends

With the aim to create a small interfering RNA (siRNA) with enhanced activity and resistance to nuclease degradation, we synthesized and evaluated the properties of the following siRNAs containing haloalkyl β-d-ribofuranosides at their 3′-dangling ends: 2,2,2-trifluoroethyl β-d-ribofuranoside, 2,2,2-trichloroethyl β-d-ribofuranoside and 2,2,2-tribromoethyl β-d-ribofuranoside. The gene silencing activities of the modified siRNAs were investigated through a dual luciferase reporter assay using HeLa cells. The highest silencing activity was observed for the trichloroethyl analog modified siRNA, which was closely followed by the trifluoroethyl and tribromoethyl analogs. The modified siRNAs were found to show increased binding affinity towards the Piwi-Argonaute-Zwille (PAZ) domain protein based on computational analysis and an experimental study. Furthermore, the RNAs modified with the analogs at their 3′-ends exhibited improved resistance to hydrolysis by a 3′-exonuclease.     

Chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy

We have developed chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3′-end for RNAi therapy. Chemically modified RNAs possessing urea/thiourea-bridged aromatic compounds instead of naturally occurring dinucleotides at the 3′-overhang region were easily prepared in good yields and were more resistant to nucleolytic hydrolysis than unmodified RNA. siRNAs containing urea or thiourea derivatives showed the desired knockdown effect. Furthermore, modified miR-143 duplexes carrying the urea/thiourea compounds in the 3′-end of each strand were able to inhibit the growth of human bladder cancer T24 cells.     

Immune activation by siRNA/liposome complexes in mice is sequence- independent: lack of a role for Toll-like receptor 3 signaling

Improvement in the pharmacokinetic properties of short interfering RNAs (siRNAs) is a prerequisite for the therapeutic application of RNA interference technology. When injected into mice as unmodified siRNAs complexed to DOTAP/Chol-based cationic liposomes, all 12 tested siRNA duplexes caused a strong induction of cytokines including interferon alpha, indicating that the immune activation by siRNA duplexes is independent of sequence context. When modified by various combinations of 2'-OMe, 2'-F, and phosphorothioate substitutions, introduction of as little as three 2'-OMe substitutions into the sense strand was sufficient to suppress immune activation by siRNA duplexes, whereas the same modifications were much less efficient at inhibiting the immune response of single stranded siRNAs. It is unlikely that Toll-like receptor 3 (TLR3) signaling is involved in immune stimulation by siRNA/liposome complexes since potent immune activation by ds siRNAs was induced in TLR3 knockout mice. Together, our results indicate that chemical modification of siRNA provides an effective means to avoid unwanted immune activation by therapeutic siRNAs. This improvement in the in vivo properties of siRNAs should greatly facilitate successful development of siRNA therapeutics.     

The effects of thiophosphate substitutions on native siRNA gene silencing

RNA mediated interference has emerged as a powerful tool in controlling gene expression in mammalian cells. We investigated the gene silencing properties of six thiophosphate substituted siRNAs (all based on a commercial luciferase medium silencer) compared to that of unmodified siRNA. We also examined the cytotoxicity and dose-response using several thiophosphate modified siRNAs with unmodified siRNA. Our results show that two thiophosphate siRNA sequences convert from medium to high silencers with the addition of four randomly placed thiophosphates. Both thiophosphate siRNAs have a statistically significant difference in luciferase gene silencing (5% and 6% activity) relative to the unmodified native medium silencer referred to as siRNA-2 (18% activity) and four other thiophosphate siRNAs that maintain their medium silencing capability. This indicates that specific thiophosphate substitutions may alter native siRNA function. Further, this shows that thiophosphate siRNAs with the same nucleotide sequence but with different sulfur modification positions have different silencing effects. Both the native siRNA and the thio siRNAs showed a concentration dependent relationship, i.e., with concentration increase, the luciferase gene silencing effect also increased. Confirming cytotoxicity experiments showed no significant changes when HeLa cells were treated with 10nM thiophosphate siRNAs over the course of several days. These results suggest that specific placement of thiophosphates could play an important role in the development of siRNAs as therapeutics by engineering in properties such as strength of binding, nuclease sensitivity, and ultimately efficacy.     

Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages

Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.     

RNA interference using boranophosphate siRNAs: structure-activity relationships

In RNA interference (RNAi), short double-stranded RNA (known as siRNA) inhibits expression from homologous genes. Clinical or pre-clinical use of siRNAs is likely to require stabilizing modifications because of the prevalence of intracellular and extracellular nucleases. In order to examine the effect of modification on siRNA efficacy and stability, we developed a new method for synthesizing stereoregular boranophosphate siRNAs. This work demonstrates that boranophosphate siRNAs are consistently more effective than siRNAs with the widely used phosphorothioate modification. Furthermore, boranophosphate siRNAs are frequently more active than native siRNA if the center of the antisense strand is not modified. Boranophosphate modification also increases siRNA potency. The finding that boranophosphate siRNAs are at least ten times more nuclease resistant than unmodified siRNAs may explain some of the positive effects of boranophosphate modification. The biochemical properties of boranophosphate siRNAs make them promising candidates for an RNAi-based therapeutic.     

Potent RNAi by short RNA triggers

RNA interference (RNAi) is a gene-silencing mechanism by which a ribonucleoprotein complex, the RNA-induced silencing complex (RISC) and a double-stranded (ds) short-interfering RNA (siRNA), targets a complementary mRNA for site-specific cleavage and subsequent degradation. While longer dsRNA are endogenously processed into 21- to 24-nucleotide (nt) siRNAs or miRNAs to induce gene silencing, RNAi studies in human cells typically use synthetic 19- to 20-nt siRNA duplexes with 2-nt overhangs at the 3′-end of both strands. Here, we report that systematic synthesis and analysis of siRNAs with deletions at the passenger and/or guide strand revealed a short RNAi trigger, 16-nt siRNA, which induces potent RNAi in human cells. Our results indicate that the minimal requirement for dsRNA to trigger RNAi is an ~42 Å A-form helix with ~1.5 helical turns. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene, suggesting that 16-nt siRNA is a more potent RNAi trigger. In vitro kinetic analysis of RNA-induced silencing complex (RISC) programmed in HeLa cells indicates that 16-nt siRNA has a higher RISC-loading capacity than 19-nt siRNA. These results suggest that RISC assembly and activation during RNAi does not necessarily require a 19-nt duplex siRNA and that 16-nt duplexes can be designed as more potent triggers to induce RNAi.     

Modified dsRNAs that are not processed by Dicer maintain potency and are incorporated into the RISC

Chemical modification of RNA duplexes can provide practical advantages for RNA interference (RNAi) triggering molecules including increased stability, safety and specificity. The impact of nucleotide modifications on Dicer processing, RISC loading and RNAi-mediated mRNA cleavage was investigated with duplexes ≥25 bp in length. It is known that dsRNAs ≥25 bp are processed by Dicer to create classic 19-bp siRNAs with 3′-end overhangs. We demonstrate that the presence of minimal modification configurations on longer RNA duplexes can block Dicer processing and result in the loading of the full-length guide strand into RISC with resultant mRNA cleavage at a defined site. These longer, modified duplexes can be highly potent gene silencers, with EC50s in the picomolar concentration range, demonstrating that Dicer processing is not required for incorporation into RISC or potent target silencing.     

Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA

The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.     

Cellular uptake and localization of a Cy3-labeled siRNA specific for the serine/threonine kinase Pim-1

A highly efficient and specific small interfering (siRNA) (PsiR4) for the serine/threonine kinase Pim-1 has been generated that silences the expression of a Pim1-green fluorescent protein (GFP) fusion gene at low nanomolar concentrations (approximately 5 nM). Only one of four siRNAs tested against Pim-1 had high potency, whereas the three other siRNAs were completely inefficient up to a concentration of 100 nM. PsiR4 was labeled with Cy3 at the 5' -end of the sense strand to investigate cellular uptake and localization in living COS-7 and F-11 cells. This modification has only minor effects on the potency of PsiR4 to inhibit Pim1-GFP. Cellular uptake of the Cy3-labeled siRNA by lipofection was observed in more than 90% of the cells and reaches a plateau 4-6 hours after transfection. Cotransfection studies with low PsiR4-Cy3 concentrations demonstrated that most cells that still expressed Pim1-GFP did not show siRNA uptake. Localization studies with PsiR4-Cy3 in the neuronal hybridoma cell line F-11 displayed a dotted, perinuclear accumulation of siRNAs. Moreover, cells with neuritelike structures contain PsiR4 in this cellular compartment.     

Practical and Reliable Synthesis of 1,2-Dideoxy-d-ribofuranose and its Application in RNAi Studies

We developed a practical and reliable method for synthesizing an abasic deoxyribonucleoside, 1,2-dideoxy-d-ribofuranose (dR^H) via elimination of nucleobase from thymidine. To synthesize oligonucleotides bearing dR^H by the standard phosphoramidite solid-phase method, dR^H was converted to the corresponding phosphoramidite derivative and linked to a solid support (controlled pore glass resin). Chemically modified small interfering RNAs (siRNAs) possessing dR^H at their 3′-overhang regions were synthesized. Introducing dR^H to the 3′-end of the antisense strand of siRNA reduced its knockdown effect.     

Competition for RISC binding predicts in vitro potency of siRNA

Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2′-O-methyl and 2′-deoxy-2′-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.     

Synthesis and properties of 4′-C-aminoalkyl-2′-fluoro-modified RNA oligomers

Synthesis and properties of double-stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) containing 4′-C-aminoethyl-2′-deoxy-2′-fluorouridine are described. Thermal denaturation studies showed that incorporation of 4′-C-aminoethyl-2′-fluoro analog improved the thermal stabilities of dsRNAs and siRNAs compared to the corresponding 4′-C-aminoethyl-2′-O-methyl analog. siRNA incorporating eight 4′-aminoethyl-2′-fluoro analogs in the passenger strand showed sufficient RNAi activity at 1?nM concentration, which was similar to that of the unmodified siRNA. Furthermore, the siRNA containing the 4′-C-aminoethyl-2′-fluoro analog exhibited high stability in a buffer containing 20% bovine serum. Forty-eight percent of the siRNA remained intact after 48?h of incubation. Thus, modification of siRNAs by the 4′-C-aminoethyl-2′-fluoro analog would be useful for the development of therapeutic siRNA molecules.