Anti-pyroptotic function of TGF-ß is suppressed by a synthetic dsRNA analogue in triple negative breast cancer cells.

Development of innovative therapeutic modalities would address an unmet clinical need in the treatment of triple negative breast cancer (TNBC). Activation of retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) such as melanoma differentiation-associated gene 5 (MDA5) and RIG-I in cancer cells is suggested to suppress tumor progression by inducing cell death. Transfection of polyI:C, a conventionally used synthetic double-stranded RNA (dsRNA) analogue that activates RLRs, has been evaluated in clinical trials. However, detailed mechanisms of tumor suppression by RLRs, especially interactions with other signaling pathways, remain elusive. Here, we showed that transfection of polyI:C suppressed transforming growth factor-ß (TGF-ß) signaling in a MDA5- and RIG-I-dependent manner. We found that suppression of TGF-ß signaling by polyI:C promoted cancer cell death, which was attenuated by forced expression of constitutively active Smad3. More detailed analysis suggested that cell death by polyI:C transfection exhibited characteristics of pyroptosis, which is distinct from apoptosis. Therapeutic efficacy of polyI:C transfection was also demonstrated using a mouse model. These results indicated that intratumor administration of polyI:C and related dsRNA analogues may be promising treatments for TNBC through inhibition of the anti-pyroptotic function of TGF-ß.

Synthesizing Fluorescently Labeled dsRNAs and sRNAs to Visualize Fungal RNA Uptake.

Fungal pathogens are responsible for severe crop losses worldwide. Defending crops against fungal disease is critical for global food security; however, most current disease management approaches rely on chemical fungicides that can leave dangerous residues in the environment. RNA interference (RNAi) is an important process through which RNA molecules target and silence complementary genes, regulating gene expression during both transcription and translation. Recently, it has been discovered that some species of fungi can efficiently take up RNAs originating from their host plant and the environment. If these RNAs are complementary to fungal genes, this can lead to the targeting and silencing of fungal genes, termed "cross-kingdom RNAi," if the RNA originated from a plant host, or "environmental RNAi," if the RNA originated from the environment. These discoveries have inspired the development of spray-induced gene silencing (SIGS), an innovative crop protection strategy involving the foliar application of RNAs which target and silence fungal virulence genes for plant protection against fungal pathogens. The effectiveness of SIGS is largely dependent on the ability of fungi to take up environmental RNAs. Here, we describe the protocols used to label and visualize RNAs which are taken up by Botrytis cinerea. This protocol could easily be adapted for use across various fungal species. Determining the efficiency of RNA uptake by a specific fungal species is a critical first step to determining if SIGS approaches could be an effective control strategy for that fungus.

[The potential of synthetic small interfering RNA-based antiviral drugs for influenza treatment].

Influenza is a worldwide public health problem. Annually, this infection affects up to 15% of the world population; and about half a million people die from this disease every year. Moreover, influenza A and B viruses tend to garner most of the attention, as these types are a major cause of the epidemics and pandemics. Although the influenza virus primarily affects the respiratory tract, it may also affect the cardiovascular and central nervous systems. Several antiviral drugs, that target various stages of viral reproduction, have been considered effective for the treatment and prevention of influenza, but some virus strains become resistant to these medications. Thus, new strategies and techniques should be developed to overcome the antiviral drug resistance. Recent studies suggest that new drugs based on RNA interference (RNAi) appear to be a promising therapeutic approach that regulates the activity of viral or cellular genes. As it is known, the RNAi is a eukaryotic gene regulatory mechanism that can be triggered by a foreign double-stranded RNA (dsRNA) and results in the cleavage of the target messenger RNA (mRNA). This review discusses the prospects, advantages, and disadvantages of using RNAi in carrying out a specific treatment for influenza infection. However, some viruses confer resistance to small interfering RNAs (siRNA) targeting viral genes. This problem can significantly reduce the effectiveness of RNAi. Therefore, applying siRNAs targeting host cell factors required for influenza virus reproduction can be a way to overcome the antiviral drug resistance.

Design of 2'-O-methyl RNA and DNA double-stranded oligonucleotides: naturally-occurring nucleotide components with strong RNA interference gene expression inhibitory activity.

Double-stranded RNAs consisting of 21-nucleotide passenger and guide strands, known as small interfering RNAs (siRNAs), can be used for the identification of gene functions and the regulation of genes involved in disease for therapeutics. The difficulty with unmodified siRNAs lies in the chemical synthesis of RNA, its degradation by RNase, the immune response derived from natural RNA, and the off-target effects mediated by the passenger strand. In this study, asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined DNAs and 2'-O-methyl RNAs, denoted as MED-siRNA, were evaluated. These modified oligonucleotides showed high RNase resistance, a reduced immune response, a highly efficient cleavage of target mRNA with binding to Argonaute 2 (Ago2) via RNA interference, and the subsequent reduction of target protein expression. These findings suggest the possibility of alternatives to unmodified siRNAs with potential use in therapeutics.

The POU factor Ventral veins lacking regulates ecdysone and juvenile hormone biosynthesis during development and reproduction of the milkweed bug, Oncopeltus fasciatus.

Recent studies have demonstrated endocrine roles for the POU domain transcription factor Ventral veins lacking (Vvl) during larval development of holometabolous insects - insects that undergo complete metamorphosis. In this study, the role of Vvl was examined in the milkweed bug, Oncopeltus fasciatus, a hemimetabolous insect. In the embryos, vvl was found to be expressed in the presumptive prothoracic glands. When vvl expression was knocked down using RNA interference (RNAi), embryos arrested their development after dorsal closure. Vvl double-stranded RNA (dsRNA)-injected nymphs failed to molt and had reduced expression of the ecdysone response gene, hormone receptor 3 (HR3), the ecdysone biosynthesis genes, disembodied and spook, and the juvenile hormone (JH) response gene, Krüppel homolog 1 (Kr-h1). Injection of 20-hydroxyecdysone rescued the molting phenotype and HR3 expression in vvl knockdown nymphs. In adults, vvl RNAi inhibited egg laying and suppressed the expression of Kr-h1 and vitellogenin in the fat body. Application of JH III or methoprene restored oviposition in vvl knockdown adults, indicating that Vvl regulates JH biosynthesis during reproduction. Thus, Vvl functions as a critical regulator of hormone biosynthesis throughout all developmental stages of O. fasciatus. Our study demonstrates that Vvl is a critical transcription factor involved in JH and ecdysteroid biosynthesis in both hemimetabolous and holometabolous insects.

Synthesis and Biological Activity of Short Interfering RNAs Having Several Consecutive Amide Internucleoside Linkages.

The success of RNA interference (RNAi) as a research tool and potential therapeutic approach has reinvigorated interest in chemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amides may be expected to improve bioavailability and cellular uptake of small interfering RNAs (siRNAs) critical for in vivo applications. In this study, we introduced up to seven consecutive amide linkages at the 3'-end of the guide strand of an siRNA duplex. Modified guide strands having four consecutive amide linkages retained high RNAi activity when paired with a passenger strand having one amide modification between its first and second nucleosides at the 5'-end. Further increase in the number of modifications decreased the RNAi activity; however, siRNAs with six and seven amide linkages still showed useful target silencing. While an siRNA duplex having nine amide linkages retained some silencing activity, the partial reduction of the negative charge did not enable passive uptake in HeLa cells. Our results suggest that further chemical modifications, in addition to amide linkages, are needed to enable cellular uptake of siRNAs in the absence of transfection agents.

Guidelines for Experiments Using Antisense Oligonucleotides and Double-Stranded RNAs.

After decades of research and development, synthetic nucleic acids are beginning to enjoy significant success in the clinic. Approved drugs have increased interest in the field, and many basic research studies have focused on synthetic nucleic acids to control the action of messenger RNA and noncoding RNAs. Unfortunately, experimental designs are often inadequate, resulting in misleading interpretation of data and unconvincing work that wastes resources and does little to advance the field. The goal of this commentary is to outline the problems facing many researchers, especially those new to the use of synthetic oligonucleotides. We describe the minimum control experiments necessary to build a strong case for real effects that are likely due to interactions at the intended molecular target. A common set of standards for preparing and judging experiments should facilitate better interpretation of data and publications that contribute positively to using synthetic nucleic acids as tools and drugs.

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.

Incorporating uracil and 5-halouracils into short peptide nucleic acids for enhanced recognition of A-U pairs in dsRNAs.

Double-stranded RNA (dsRNA) structures form triplexes and RNA-protein complexes through binding to single-stranded RNA (ssRNA) regions and proteins, respectively, for diverse biological functions. Hence, targeting dsRNAs through major-groove triplex formation is a promising strategy for the development of chemical probes and potential therapeutics. Short (e.g., 6-10 mer) chemically-modified Peptide Nucleic Acids (PNAs) have been developed that bind to dsRNAs sequence specifically at physiological conditions. For example, a PNA incorporating a modified base thio-pseudoisocytosine (L) has an enhanced recognition of a G-C pair in an RNA duplex through major-groove L·G-C base triple formation at physiological pH, with reduced pH dependence as observed for C+·G-C base triple formation. Currently, an unmodified T base is often incorporated into PNAs to recognize a Watson-Crick A-U pair through major-groove T·A-U base triple formation. A substitution of the 5-methyl group in T by hydrogen and halogen atoms (F, Cl, Br, and I) causes a decrease of the pKa of N3 nitrogen atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions. Here, we synthesized a series of PNAs incorporating uracil and halouracils, followed by binding studies by non-denaturing polyacrylamide gel electrophoresis, circular dichroism, and thermal melting. Our results suggest that replacing T with uracil and halouracils may enhance the recognition of an A-U pair by PNA·RNA2 triplex formation in a sequence-dependent manner, underscoring the importance of local stacking interactions. Incorporating bromouracils and chlorouracils into a PNA results in a significantly reduced pH dependence of triplex formation even for PNAs containing C bases, likely due to an upshift of the apparent pKa of N3 atoms of C bases. Thus, halogenation and other chemical modifications may be utilized to enhance hydrogen bonding of the adjacent base triples and thus triplex formation. Furthermore, our experimental and computational modelling data suggest that PNA·RNA2 triplexes may be stabilized by incorporating a BrUL step but not an LBrU step, in dsRNA-binding PNAs.

Synergistic effects of stellated fibrous mesoporous silica and synthetic dsRNA analogues for cancer immunotherapy.

Stellated fibrous mesoporous silica nanospheres significantly improve the cellular uptake of cancer antigen and the maturation of bone marrow derived dendritic cells in vitro. Moreover, the combination of poly(I:C) with stellated fibrous MS nanospheres markedly decreases the necessary dose of poly(I:C) for anti-tumor immunity, and thus opens new opportunities for the future clinical application of poly(I:C) in cancer immunotherapy.

Induction of RNAi Responses by Short Left-Handed Hairpin RNAi Triggers.

Small double-stranded, left-handed hairpin (LHP) RNAs containing a 5'-guide-loop-passenger-3' structure induce RNAi responses by a poorly understood mechanism. To explore LHPs, we synthesized fully 2'-modified LHP RNAs targeting multiple genes and found all to induce robust RNAi responses. Deletion of the loop and nucleotides at the 5'-end of the equivalent passenger strand resulted in a smaller LHP that still induced strong RNAi responses. Surprisingly, progressive deletion of up to 10 nucleotides from the 3'-end of the guide strand resulted in a 32mer LHP capable of inducing robust RNAi responses. However, further guide strand deletion inhibited LHP activity, thereby defining the minimal length guide targeting length to 13 nucleotides. To dissect LHP processing, we examined LHP species that coimmunoprecipitated with Argonaute 2 (Ago2), the catalytic core of RNA-induced silencing complex, and found that the Ago2-associated processed LHP species was of a length that correlated with Ago2 cleavage of the passenger strand. Placement of a blocking 2'-OMe blocking modification at the LHP predicted Ago2 cleavage site resulted in an intact LHP loaded into Ago2 and no RNAi response. Taken together, these data argue that in the absence of a substantial loop, this novel class of small LHP RNAs enters the RNAi pathway by a Dicer-independent mechanism that involves Ago2 cleavage and results in an extended guide strand. This work establishes LHPs as an alternative RNAi trigger that can be produced from a single synthesis for potential use as an RNAi therapeutic.

Synthesis of native-like crosslinked duplex RNA and study of its properties.

A variety of enzymes have been found to interact with double-stranded RNA (dsRNA) in order to carry out its functions. We have endeavored to prepare the covalently crosslinked native-like duplex RNA, which could be useful for biochemical studies and RNA nanotechnology. In this study, the interstrand covalently linked duplex RNA was formed by a crosslinking reaction between vinylpurine (VP) and the target cytosine or uracil in RNA. We measured melting temperatures and CD spectra to identify the properties of the VP crosslinked duplex RNA. The crosslinking formation increased the thermodynamic stability without disturbing the natural conformation of dsRNA. In addition, a competitive binding experiment with the duplex RNA binding enzyme, ADAR2, showed the crosslinked dsRNA bound the protein with nearly the same binding affinity as the natural dsRNA, confirming that it has finely preserved the natural traits of duplex RNA.

Highly Ordered Pyrene π-Stacks on an RNA Duplex.

The syntheses of 2'-O-(pyren-1-ylmethyl)uridine phosphoramidite, 2'-O-(pyren-1-ylmethyl)adenosine phosphoramidite, and multiple pyrene-attached oligo-RNAs are described in this unit. The 2'-O-(pyren-1-ylmethyl)nucleosides are converted into the corresponding 2'-O-(pyren-1-ylmethyl)nucleoside 3'-phosphoramidites, which can be incorporated into the specific position of oligo-RNAs by solid-phase oligonucleotide synthesis. The multiple pyrene-attached oligo-RNA forms an A-form duplex with a complementary multiple pyrene-attached oligo-RNA; the pyrenes are associated with π-stacking along the outside of the duplex.

Non-invasive delivery of dsGST is lethal to the sweet potato whitefly, Bemisia tabaci (G.) (Hemiptera: Aleyrodidae).

The sweet potato whitefly, Bemisia tabaci (G.) biotype B (Hemiptera: Aleyrodidae), is one of the most economically important pest, by being a dreaded vector of Geminiviruses, and also causes direct damage to the crops by sucking phloem sap. Glutathione S-transferase (GST) is a large family of multifunctional enzymes that play pivotal roles in the detoxification of secondary allelochemical produced by the host plants and in insecticide resistance, thus regulates insect growth and development. The objective of this study is to show the potential of RNA interference (RNAi) in the management of B. tabaci. RNAi is a sequence-specific gene silencing mechanism induced by double-stranded RNA (dsRNA) which holds tremendous potential in pest management. In this regard, we sequenced the GST from B. tabaci and synthesized approximately 500-bp dsRNA from the above and delivered through diet to B. tabaci. Real-time quantitative PCR (RT-qPCR) showed that continuous application of dsGST at 1.0, 0.5, and 0.25 µg/µl reduced mRNA expression levels for BtGST by 77.43, 64.86, and 52.95 % which resulted in mortality by 77, 59, and 40 %, respectively, after 72 h of application. Disruption of BtGST expression will enable the development of novel strategies in pest management and functional analysis of vital genes in B. tabaci.

Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α.

Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). Their abundances vary in context-dependent fashion spatiotemporarily and there is mounting evidence of regulatory interplay between them. Here, we introduce chemically synthesized pre-miRNAs (syn-pre-miRNAs) as a general class of accessible, easily transfectable mimics of pre-miRNAs. These are RNA hairpins, identical in sequence to natural pre-miRNAs. They differ from commercially available miRNA mimics through their complete hairpin structure, including any regulatory elements in their terminal-loop regions and their potential to introduce both strands into RISC. They are distinguished from transcribed pre-miRNAs by their terminal 5' hydroxyl groups and their precisely defined terminal nucleotides. We demonstrate with several examples how they fully recapitulate the properties of pre-miRNAs, including their processing by Dicer into functionally active 5p; and 3p-derived mature miRNAs. We use syn-pre-miRNAs to show that miR-34a uses its 5p and 3p miRNAs in two pathways: apoptosis during TGF-ß signaling, where SIRT1 and SP4 are suppressed by miR-34a-5p and miR-34a-3p, respectively; and the lipopolysaccharide (LPS)-activation of primary human monocyte-derived macrophages, where TNF (TNFα) is suppressed by miR-34a-5p indirectly and miR-34a-3p directly. Our results add to growing evidence that the use of both arms of a miRNA may be a widely used mechanism. We further suggest that syn-pre-miRNAs are ideal and affordable tools to investigate these mechanisms.

Long double-stranded multiplex siRNAs for dual genes silencing.

Simultaneous suppression of multiple oncogenes is an attractive strategy to treat cancers. Herein we present a series of long double-stranded multiplex small interfering RNAs (multi-siRNAs) that is suitable for dual genes silencing through a sequence-specific RNA interference process without inducing significant immune responses. A gap feature structurally designed in either of the nucleotide strands of the multi-siRNAs was proved to be essential toward silencing target genes and avoiding immune responses. Furthermore, the silencing effect of multi-siRNAs against SURVIVIN and BCL-2 genes was shown to be effective and resulted in up-regulation of caspase-3 related apoptosis and, in turn, inhibition of bladder cancer cell proliferation. Our observation suggested that the rationally designed multi-siRNAs would have great potential for therapeutic siRNA design.

Quantitation of the dynamic profiles of the innate immune response using multiplex selected reaction monitoring-mass spectrometry.

The innate immune response (IIR) is a coordinated intracellular signaling network activated by the presence of pathogen-associated molecular patterns that limits pathogen spread and induces adaptive immunity. Although the precise temporal activation of the various arms of the IIR is a critical factor in the outcome of a disease, currently there are no quantitative multiplex methods for its measurement. In this study, we investigate the temporal activation pattern of the IIR in response to intracellular double-stranded RNA stimulation using a quantitative 10-plex stable isotope dilution-selected reaction monitoring-MS assay. We were able to observe rapid activation of both NF-κB and IRF3 signaling arms, with IRF3 demonstrating a transient response, whereas NF-κB underwent a delayed secondary amplification phase. Our measurements of the NF-κB-IκBα negative feedback loop indicate that about 20% of IκBα in the unstimulated cell is located within the nucleus and represents a population that is rapidly degraded in response to double-stranded RNA. Later in the time course of stimulation, the nuclear IκBα pool is repopulated first prior to its cytoplasmic accumulation. Examination of the IRF3 pathway components shows that double-stranded RNA induces initial consumption of the RIG-I PRR and the IRF3 kinase (TBK1). Stable isotope dilution-selected reaction monitoring-MS measurements after siRNA-mediated IRF3 or RelA knockdown suggests that a low nuclear threshold of NF-κB is required for inducing target gene expression, and that there is cross-inhibition of the NF-κB and IRF3 signaling arms. Finally, we were able to measure delayed noncanonical NF-κB activation by quantifying the abundance of the processed (52 kDa) NF-κB2 subunit in the nucleus. We conclude that quantitative proteomics measurement of the individual signaling arms of the IIR in response to system perturbations is significantly enabled by stable isotope dilution-selected reaction monitoring-MS-based quantification, and that this technique will reveal novel insights into the dynamics and connectivity of the IIR.

Silencing of genes and alleles by RNAi in Anopheles gambiae.

Anopheles gambiae mosquitoes are the major vectors of human malaria parasites. However, mosquitoes are not passive hosts for parasites, actively limiting their development in vivo. Our current understanding of the mosquito antiparasitic response is mostly based on the phenotypic analysis of gene knockdowns obtained by RNA interference (RNAi), through the injection or transfection of long dsRNAs in adult mosquitoes or cultured cells, respectively. Recently, RNAi has been extended to silence specifically one allele of a given gene in a heterozygous context, thus allowing to compare the contribution of different alleles to a phenotype in the same genetic background.

Analysis of Legionella infection using RNAi in Drosophila cells.

RNA interference (RNAi) is the process of specific gene silencing by the use of double-stranded RNA (dsRNA). In cultured Drosophila cells, RNAi methodologies are well established and easily executed: dsRNA, when added to the cell culture medium, is efficiently internalized by the cells and, through the activity of endogenous processing machinery, targets the specified mRNA for degradation resulting in reduced levels of its encoded protein. This technique has proven very useful in studying the role of host genes during Legionella pneumophila infections, as it allows the effect of host factor depletion on intracellular growth of the bacterium to be examined. In this chapter we present the methods commonly used in our laboratory to study intracellular growth of L. pneumophila using dsRNA in Drosophila cells.

Uptake of extracellular double-stranded RNA by SID-2.

Ingested dsRNAs trigger RNA interference (RNAi) in many invertebrates, including the nematode Caenorhabditis elegans. Here we show that the C. elegans apical intestinal membrane protein SID-2 is required in C. elegans for the import of ingested dsRNA and that, when expressed in Drosophila S2 cells, SID-2 enables the uptake of dsRNAs. SID-2-dependent dsRNA transport requires an acidic extracellular environment and is selective for dsRNAs with at least 50 base pairs. Through structure-function analysis, we identify several SID-2 regions required for this activity, including three extracellular, positively charged histidines. Finally, we find that SID-2-dependent transport is inhibited by drugs that interfere with vesicle transport. Therefore, we propose that environmental dsRNAs are imported from the acidic intestinal lumen by SID-2 via endocytosis and are released from internalized vesicles in a secondary step mediated by the dsRNA channel SID-1. Similar multistep mechanisms may underlie the widespread observations of environmental RNAi.