Variations of the 3' protruding ends in synthetic short interfering RNA (siRNA) tested by microinjection in Drosophila embryos

Short interfering RNAs (siRNAs) are the processing product originating from long double-stranded RNAs (dsRNAs) that are cleaved by the RNase III-like ribonuclease Dicer. As siRNAs mediate cleavage of specific single-stranded target RNAs, they are essential intermediates of RNA interference (RNAi). When applied in synthetic form, siRNAs likewise can induce the silencing process in the absence of long dsRNAs. Here, we tested variations of a conventional synthetic siRNA that had been used successfully to silence the Drosophila notch gene. The variants had two 3 ' -terminal deoxynucleotides in their protruding single-stranded ends. In one case, the deoxynulceotides would match to the notch mRNA, whereas the other variant had nonmatching deoxy-T residues, representing a widely used siRNA design. siRNAs with different combinations of sense and antisense strands were injected into Drosophila embryos at two different concentrations. We found that the all-ribonucleotide siRNA gave the best inhibition of notch expression. The combination of two modified strands with 3 ' -terminal deoxynucleotides was effective, but if combined with a sense or antisense ribostrand, the efficacy dropped. The siRNAs with nonmatching 3 ' -terminal TT residues showed a reduced silencing potential, which became evident at low concentration. An siRNA with a nonmatching 3 ' -terminal ribonucleotide in the antisense strand retained most of its silencing potential in accordance with the hypothesis that primer extension for generation of ssRNA from single-stranded mRNA does not operate in Drosophila.     

Functional comparison of single- and double-stranded siRNAs in mammalian cells

The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double- and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them.     

Delivery of siRNA using hyaluronic acid-guided nanoparticles for downregulation of CXCR4

In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 μg mL⁻¹ (except AuPEI NPs) in vitro, cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.     

Focusing on RISC assembly in mammalian cells

RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5′ end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5′ end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi.     

Reducible siRNA dimeric conjugates for efficient cellular uptake and gene silencing

In this study, dimerized siRNAs linked by a cleavable disulfide bond were synthesized for efficient intracellular delivery and gene silencing. The reducible dimerized siRNAs showed far enhanced complexation behaviors with cationic polymers as compared to monomeric siRNA at the same N/P ratio, as demonstrated by microscopic techniques and gel characterization. Dimerized siRNAs targeting green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) were complexed with linear polyethylenimine (LPEI), and treated to various cell lines to examine gene transfection efficiencies. In comparison to monomer siRNA/LPEI complexes, dimeric siRNA/LPEI complexes showed greatly enhanced cellular uptake and gene silencing effects in vitro. These results were mainly due to the higher charge density and promoted chain flexibility of the dimerized siRNAs, providing more compact and stable siRNA complexes. In addition, the conjugation strategy of reducible siRNA dimers was further extended: poly(ethylene glycol) (PEG)-modified dimerized siRNAs and heterodimers of siRNAs targeting two different genes (e.g., GFP and VEGF) were synthesized, and their gene silencing efficiencies were characterized. The dimerized siRNA complex system holds great potential for in vivo systemic gene therapy.     

Single-stranded antisense siRNAs guide target RNA cleavage in RNAi

Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.     

Targeted delivery of siRNA to hepatocytes and hepatic stellate cells by bioconjugation

Previously, we successfully conjugated galactosylated poly(ethylene glycol) (Gal-PEG) to oligonucleotides (ODNs) via an acid labile ester linker (Zhu et al., Bioconjugate Chem. 2008, 19, 290-8). In this study, sense strands of siRNA were conjugated to Gal-PEG and mannose 6-phosphate poly(ethylene glycol) (M6P-PEG) for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells (HSCs), respectively. These siRNA conjugates were purified by ion exchange chromatography and verified by gel retardation assay. To evaluate their RNAi functions, the validated siRNA duplexes targeting firefly luciferase and transforming growth factor beta 1 (TGF-β1) mRNA were conjugated to Gal-PEG and M6P-PEG, and their gene silencing efficiencies were determined after transfection into HepG2 and HSC-T6 cells. The disulfide bond between PEG and siRNA was cleaved by dithiothreitol, leading to the release of intact siRNA. Both Gal-PEG-siRNA and M6P-PEG-siRNA conjugates could silence luciferase gene expression by about 40% without any transfection reagents, while the gene silencing effects reached more than 98% with the help of cationic liposomes at the same dose. Conjugation of TGF-β1 siRNA with Gal-PEG and M6P-PEG could silence endogenous TGF-β1 gene expression as well. In conclusion, these siRNA conjugates have the potential for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells for efficient gene silencing in vivo.     

Sequence,chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing

Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5'-end of the sense strand, only 21-29 bp hairpins were highly active.     

Poly-2'-DNP-RNAs with enhanced efficacy for inhibiting cancer cell growth

It is often believed that small interfering RNA (siRNA) is at least 10-fold more effective than the single-stranded antisense oligonucleotide for silencing the same target gene in the same cells. In view of the recent discovery that the RNA-induced silencing complex (RISC) contains only a single-stranded RNA (ssRNA) molecule and can be reconstituted using single-stranded antisense RNA, such a large difference in efficacy seems puzzling. One possible reason is that hybridization protects siRNA from hydrolysis by endogenous RNase activity until it is incorporated in the RISC, whereas ssRNA is rapidly hydrolyzed. Because the single-stranded poly-2'-O-(2,4-dinitrophenyl)-RNA (DNP-ssRNA) is both RNase resistant and membrane permeable, we synthesized homologous native siRNAs, DNP-siRNAs, native ssRNAs, and DNP-ssRNAs and made a comparative study of their efficacies for inhibiting the growth of two cancer cell lines with different overexpressed target genes under equivalent experimental conditions. It was found that the efficacy of antisense DNP-ssRNA is higher than that of the corresponding siRNA and that the efficacy of native siRNA for inhibiting cell growth can also be enhanced from 2-fold to 6-fold by replacing the native strands of RNA in siRNA with homologous DNP-RNA. Thermal denaturation data show that the hybridization affinity of the DNP-RNA/RNA duplex is higher than that of the native RNA/RNA duplex. Western blotting analysis of A549 cells treated with antisense DNP-ssRNAs containing single mismatching bases shows that the gene silencing by antisense DNP-ssRNA is as sequence specific as that by siRNA. The observed large enhancement of inhibition efficacy of native RNAs by DNP derivatization should be advantageous for both gene silencing studies and therapeutic applications.     

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.     

Self-crosslinked and reducible fusogenic peptides for intracellular delivery of siRNA

A novel self-crosslinked and reducible peptide was synthesized for stable formation of nanoscale complexes with an siRNA-PEG conjugate to enhance transfection efficiency in serum containing condition without compromising cytotoxicity. A fusogenic peptide, KALA, with two cysteine residues at both terminal ends was crosslinked via disulfide linkages under mild DMSO oxidation condition. The reducible crosslinked KALA (cl-KALA) was used to form nano-complexes with green fluorescent protein (GFP) siRNA. Size and morphology of various polyelectrolyte complexes formulated with KALA and cl-KALA were comparatively analyzed. cl-KALA exhibited more reduced cell cytotoxicity and formed more stable and compact polyelectrolyte complexes with siRNA, compared with naked KALA and polyethylenimine (PEI), probably because of its increased charge density. The extent of gene silencing was quantitatively evaluated using MDA-MB-435 cells. cl-KALA/siRNA complexes showed comparable gene silencing efficiency with those of cytotoxic PEI. In a serum containing medium, cl-KALA/siRNA-PEG conjugate complexes exhibited superior gene inhibition because of the shielding effect of PEG on the surface. The formulation based on the self-crosslinked fusogenic peptide could be used as a biocompatible and efficient nonviral carrier for siRNA delivery. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 881-888, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5'-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.     

Intracellular small interfering RNA delivery using genetically engineered double‐stranded RNA binding protein domain

Background

A variety of synthetic carriers, such as cationic polymers and lipids, have been used as nonviral carriers for small interfering RNA (siRNA) delivery. Although siRNA polyplexes and lipoplexes exhibited good gene silencing efficiencies, they often showed serious cytotoxicities, which are not useful for clinical applications. A double‐stranded RNA binding cellular protein with highly specific siRNA binding property and noncytotoxicity was used for siRNA delivery.

Methods

A double‐stranded RNA binding domain (dsRBD) of human double‐stranded RNA activated protein kinase R was genetically produced and utilized to complex siRNA for intracellular delivery. For characterization of the siRNA/dsRBD complexes, decomplexation assay and RNase protection assay were performed. Cytotoxicity and target gene inhibition ability were also examined using human carcinoma cell lines.

Results

The recombinantly produced polypeptide dsRBD exhibited its inherent binding activity for siRNA without sequence specificity, and the siRNA/dsRBD complexes protected siRNA from degradation by ribonucleases. Green fluorescent protein (GFP) siRNA/dsRBD complexes showed prominent down‐regulation of a target GFP gene, when an endosomal escape function was supplemented by addition of a fusogenic peptide, KALA, in the formulation.

Conclusions

The results suggest that dsRBD‐based protein carriers could be successfully applied for a wide range of therapeutic siRNAs for intracellular gene inhibition without showing any cytotoxicity. Copyright © 2009 John Wiley & Sons, Ltd.     

Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA

Short interfering RNAs (siRNAs) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function and are also being developed for therapeutic applications. Many nucleic acids, including double- (dsRNA) and single-stranded RNA (ssRNA), can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system. This could significantly influence the in vivo application of siRNA owing to off-target effects and toxicities associated with immune stimulation. Here we report that synthetic siRNAs formulated in nonviral delivery vehicles can be potent inducers of interferons and inflammatory cytokines both in vivo in mice and in vitro in human blood. The immunostimulatory activity of formulated siRNAs and the associated toxicities are dependent on the nucleotide sequence. We have identified putative immunostimulatory motifs that have allowed the design of siRNAs that can mediate RNAi but induce minimal immune activation.     

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.     

Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses

RNA silencing is an evolutionarily conserved surveillance system that occurs in a broad range of eukaryotic organisms. In plants, RNA silencing acts as an antiviral system; thus, successful virus infection requires suppression of gene silencing. A number of viral suppressors have been identified so far; however, the molecular bases of silencing suppression are still poorly understood. Here we show that p19 of Cymbidium ringspot virus (CymRSV) inhibits RNA silencing via its small RNA-binding activity in vivo. Small RNAs bound by p19 in planta are bona fide double-stranded siRNAs and they are silencing competent in the in vitro RNA-silencing system. p19 also suppresses RNA silencing in the heterologous Drosophila in vitro system by preventing siRNA incorporation into RISC. During CymRSV infection, p19 markedly diminishes the amount of free siRNA in cells by forming p19-siRNA complexes, thus making siRNAs inaccessible for effector complexes of RNA-silencing machinery. Furthermore, the obtained results also suggest that the p19-mediated sequestration of siRNAs in virus-infected cells blocks the spread of the mobile, systemic signal of RNA silencing.