Small Antisense DNA-Based Gene Silencing Enables Cell-Free Bacteriophage Manipulation and Genome Replication.

Cell-free systems allow interference with gene expression processes without requiring elaborate genetic engineering procedures. This makes it ideally suited for rapid prototyping of synthetic biological parts. Inspired by nature's strategies for the control of gene expression via short antisense RNA molecules, we here investigated the use of small DNA (sDNA) for translational inhibition in the context of cell-free protein expression. We designed sDNA molecules to be complementary to the ribosome binding site (RBS) and the downstream coding sequence of targeted mRNA molecules. Depending on sDNA concentration and the promoter used for transcription of the mRNA, this resulted in a reduction of gene expression of targeted genes by up to 50-fold. We applied the cell-free sDNA technique (CF-sDNA) to modulate cell-free gene expression from the native T7 phage genome by suppressing the production of the major capsid protein of the phage. This resulted in a reduced phage titer, but at the same time drastically improved cell-free replication of the phage genome, which we utilized to amplify the T7 genome by more than 15 000-fold in a droplet-based serial dilution experiment. Our simple antisense sDNA approach extends the possibilities to exert translational control in cell-free expression systems, which should prove useful for cell-free prototyping of native phage genomes and also cell-free phage manipulation.

Development of a bioanalytical method for an antisense therapeutic using high-resolution mass spectrometry.

Background: Ion-pairing reverse-phase LC coupled with high-resolution mass spectrometry (IP-LC/HRMS) has gained attention in oligonucleotide therapeutic bioanalyses owing to its high sensitivity and selectivity. However, optimization and validation of IP-LC/HRMS-based methods are rare. The objective of this study is the development of a sensitive and reproducible IP-LC/HRMS-based bioanalytical method using clinically approved mipomersen as a model for antisense oligonucleotides. Materials & methods/results: Mipomersen was extracted from rat plasma using Clarity OTX SPE and quantified by IP-LC/HRMS. The calibration range was 0.5-250.0 ng/ml. The developed method met the general regulatory criteria for accuracy, precision, carry-over, selectivity, matrix effect and dilution integrity. Conclusion: A highly sensitive and reliable method for mipomersen measurement with potential antisense oligonucleotide bioanalysis applications has been developed.

Disulfide-Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA.

Development of intracellular delivery methods for antisense DNA and siRNA is important. Previously reported methods using liposomes or receptor-ligands take several hours or more to deliver oligonucleotides to the cytoplasm due to their retention in endosomes. Oligonucleotides modified with low molecular weight disulfide units at a terminus reach the cytoplasm 10 minutes after administration to cultured cells. This rapid cytoplasmic internalization of disulfide-modified oligonucleotides suggests the existence of an uptake pathway other than endocytosis. Mechanistic analysis revealed that the modified oligonucleotides are efficiently internalized into the cytoplasm through disulfide exchange reactions with the thiol groups on the cellular surface. This approach solves several critical problems with the currently available methods for enhancing cellular uptake of oligonucleotides and may be an effective approach in the medicinal application of antisense DNA and siRNA.

A Potential Role for the STXBP5-AS1 Gene in Adult ADHD Symptoms.

We aimed to detect Attention-deficit/hyperactivity (ADHD) risk-conferring genes in adults. In children, ADHD is characterized by age-inappropriate levels of inattention and/or hyperactivity-impulsivity and may persists into adulthood. Childhood and adulthood ADHD are heritable, and are thought to represent the clinical extreme of a continuous distribution of ADHD symptoms in the general population. We aimed to leverage the power of studies of quantitative ADHD symptoms in adults who were genotyped. Within the SAGA (Study of ADHD trait genetics in adults) consortium, we estimated the single nucleotide polymorphism (SNP)-based heritability of quantitative self-reported ADHD symptoms and carried out a genome-wide association meta-analysis in nine adult population-based and case-only cohorts of adults. A total of n = 14,689 individuals were included. In two of the SAGA cohorts we found a significant SNP-based heritability for self-rated ADHD symptom scores of respectively 15% (n = 3656) and 30% (n = 1841). The top hit of the genome-wide meta-analysis (SNP rs12661753; p-value = 3.02 × 10-7) was present in the long non-coding RNA gene STXBP5-AS1. This association was also observed in a meta-analysis of childhood ADHD symptom scores in eight population-based pediatric cohorts from the Early Genetics and Lifecourse Epidemiology (EAGLE) ADHD consortium (n = 14,776). Genome-wide meta-analysis of the SAGA and EAGLE data (n = 29,465) increased the strength of the association with the SNP rs12661753. In human HEK293 cells, expression of STXBP5-AS1 enhanced the expression of a reporter construct of STXBP5, a gene known to be involved in "SNAP" (Soluble NSF attachment protein) Receptor" (SNARE) complex formation. In mouse strains featuring different levels of impulsivity, transcript levels in the prefrontal cortex of the mouse ortholog Gm28905 strongly correlated negatively with motor impulsivity as measured in the five choice serial reaction time task (r2 = - 0.61; p = 0.004). Our results are consistent with an effect of the STXBP5-AS1 gene on ADHD symptom scores distribution and point to a possible biological mechanism, other than antisense RNA inhibition, involved in ADHD-related impulsivity levels.

Complex consisting of antisense DNA and ß-glucan promotes internalization into cell through Dectin-1 and hybridizes with target mRNA in cytosol.

Antisense oligonucleotides (AS-ODNs) hybridize with specific mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. We previously demonstrated that the ß-glucan schizophyllan (SPG) can form a complex with AS-ODNs with attached dA40 (AS-ODNs/SPG), and this complex can be incorporated into cells, such as macrophages and dendritic cells, expressing the ß-glucan receptor Dectin-1. We have achieved efficient gene silencing in animal models, but the uptake mechanism and intracellular distribution are unclear. In this study, we prepared the complex consisting of SPG and AS-ODNs (AS014) for Y-box binding protein-1 (YB-1). After treatment with endocytosis inhibitor Pitstop 2 and small interfering RNA targeting Dectin-1, we found that AS014/SPG complexes are incorporated into cells by Dectin-1-mediated endocytosis and inhibit cell growth in a Dectin-1 expression level-dependent manner. After treatment with AS014/SPG complexes, we separated the cell lysate into endosomal and cytoplasmic components by ultracentrifugation and directly determined the distribution of AS014 by reverse transcription PCR using AS014 ODNs as a template or a reverse transcription primer. In the cytoplasm, AS014 clearly hybridized with YB-1 mRNAs. This is the first demonstration of the distinct distribution of the complex in cells. These results could facilitate the clinical application of the complex.

Cytosolic Genomic DNA functions as a Natural Antisense.

Stress conditions such as UV irradiation, exposure to genotoxic agents, stalled DNA replication, and even tumors trigger the release of cytosolic genomic DNA (cgDNA). Classically, cgDNA induces interferon response via its binding to proteins such as STING. In this study, we found previously reported cgDNA (cg721) exists in the cytosol of the mouse cell lines, cultured under no stress conditions. The overexpression of cg721 suppressed the complementary RNA expression using strand selection and knockdown of DNA/RNA hybrid R-loop removing enzyme RNase H and three prime repair exonuclease 1 TREX1 increased the expression levels of cg721 and thus, inhibited the target Naa40 transcript, as well as protein expression, with a phenotypic effect. In addition, cgDNA was incorporated into extracellular vesicles (EVs), and the EV-derived cg721 inhibited gene expression of the acceptor cells. Thus, our findings suggest that cg721 functions as a natural antisense DNA and play a role in cell-to-cell gene regulation once it secreted outside the cell as EVs.

mbtJ: an iron stress-induced acetyl hydrolase/esterase of Mycobacterium tuberculosis helps bacteria to survive during iron stress.

AIM: mbtJ from Mycobacterium tuberculosis H37Rv is a member of mbt A-J operon required for mycobactin biogenesis. MATERIALS & METHODS: The esterase/acetyl-hydrolase activity of mbtJ was determined by pNP-esters/native-PAGE and expression under iron stress by quantitative-PCR. Effect of gene on growth/survival of Mycobacterium was studied using antisense. Its effect on morphology, growth/infection was studied in Mycobacterium smegmatis. RESULTS: It showed acetyl hydrolase/esterase activity at pH 8.0 and 50°C with pNP-butyrate. Its expression was upregulated under iron stress. The antisense inhibited the survival of bacterium during iron stress. Expression of mbtJ changed colony morphology and enhanced the growth/infection in M. smegmatis. CONCLUSION: mbtJ, an acetyl-hydrolase/esterase, enhanced the survival of M. tuberculosis under iron stress, affected the growth/infection efficiency in M. smegmatis, suggesting its pivotal role in the intracellular survival of bacterium.

Specific RNP capture with antisense LNA/DNA mixmers.

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute.

Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5' end of the guide strand. This explains why in vivo TtAgo is preferentially loaded with guides with a 5' end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.

Antisense locked nucleic acids targeting agrA inhibit quorum sensing and pathogenesis of community-associated methicillin-resistant Staphylococcus aureus.

AIM: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is commonly associated with nonnosocomial skin and soft tissue infections due to its virulence, which is mainly controlled by the accessory gene regulator (agr) quorum sensing (QS) system. In this study (KFF)3 K peptide-conjugated locked nucleic acids (PLNAs) targeting agrA mRNA were developed to inhibit agr activity and arrest the pathogenicity of CA-MRSA. METHODS AND RESULTS: Two PLNAs were designed, and synthesized, after predicting the secondary structure of agrA mRNA. The influence on bacterial growth was tested using a growth curve assay. RT-qPCR, haemolysis assay, lactate dehydrogenase release assay and chemotaxis assay were used to evaluate the effects of the PLNAs on inhibiting agr QS. A mouse skin infection model was employed to test the protective effect of the PLNAs in vivo. None of the PLNAs were found to be bacteriostatic or bactericidal in vitro. However, one PLNA, PLNA34, showed strong ability to suppress expression of agrA and the effector molecule RNAIII in USA300 LAC strain. Furthermore, PLNA34 inhibited the expression of virulence genes that are upregulated by agr, including hla, psmα, psmß and pvl. The haemolytic activity of the supernatants from PLNA34-treated bacteria was also dramatically reduced, as well as the capacity to lyse and recruit neutrophils. Moreover, PLNA34 showed high levels of protection in the CA-MRSA mouse skin infection model. CONCLUSIONS: The anti-agrA PLNA34 can effectively inhibit the agr QS and suppress CA-MRSA pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: agrA is a promising target for the development of antisense oligonucleotides to block agr QS.

Natural antisense transcripts are linked to the modulation of mitochondrial function and teliospore dormancy in Ustilago maydis.

The basidiomycete smut fungus Ustilago maydis causes common smut of corn. This disease is spread through the production of teliospores, which are thick-walled dormant structures characterized by low rates of respiration and metabolism. Teliospores are formed when the fungus grows within the plant, and the morphological steps involved in their formation have been described, but the molecular events leading to dormancy are not known. In U. maydis, natural antisense transcripts (NATs) can function to alter gene expression and many NATs have increased levels in the teliospore. One such NAT is as-ssm1 which is complementary to the gene for the mitochondrial seryl-tRNA synthetase (ssm1), an enzyme important to mitochondrial function. The disruption of ssm1 leads to cell lysis, indicating it is also essential for cellular viability. To assess the function of as-ssm1, it was ectopically expressed in haploid cells, where it is not normally present. This expression led to reductions in growth rate, virulence, mitochondrial membrane potential and oxygen consumption. It also resulted in the formation of as-ssm1/ssm1 double-stranded RNA and increased ssm1 transcript levels, but no change in Ssm1 protein levels was detected. Together, these findings suggest a role for as-ssm1 in facilitating teliospore dormancy through dsRNA formation and reduction of mitochondrial function.

A general strategy to inhibiting viral -1 frameshifting based on upstream attenuation duplex formation.

Viral -1 programmed ribosomal frameshifting (PRF) as a potential antiviral target has attracted interest because many human viral pathogens, including human immunodeficiency virus (HIV) and coronaviruses, rely on -1 PRF for optimal propagation. Efficient eukaryotic -1 PRF requires an optimally placed stimulator structure downstream of the frameshifting site and different strategies targeting viral -1 PRF stimulators have been developed. However, accessing particular -1 PRF stimulator information represents a bottle-neck in combating the emerging epidemic viral pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV). Recently, an RNA hairpin upstream of frameshifting site was shown to act as a cis-element to attenuate -1 PRF with mechanism unknown. Here, we show that an upstream duplex formed in-trans, by annealing an antisense to its complementary mRNA sequence upstream of frameshifting site, can replace an upstream hairpin to attenuate -1 PRF efficiently. This finding indicates that the formation of a proximal upstream duplex is the main determining factor responsible for -1 PRF attenuation and provides mechanistic insight. Additionally, the antisense-mediated upstream duplex approach downregulates -1 PRF stimulated by distinct -1 PRF stimulators, including those of MERS-CoV, suggesting its general application potential as a robust means to evaluating viral -1 PRF inhibition as soon as the sequence information of an emerging human coronavirus is available.

Genetic manipulation of RPS5 gene expression modulates the initiation of commitment of MEL cells to erythroid maturation: Implications in understanding ribosomopathies.

Impairment of ribosome biogenesis contributes to the molecular pathophysiology of ribosomopathies by deregulating cell-lineage specific proliferation, differentiation and apoptosis decisions of haematopoietic progenitor cells. Here, using pro-erythroblast-like murine erythroleukemia (MEL) cells, a model system of erythroid maturation, we aimed to investigate whether genetic manipulation of RPS5 expression affects the capacity of cells to grow and differentiate in culture. Parental MEL cells stably transfected with full length RPS5 cDNA in sense (MEL-C14 culture) or antisense (MEL-antisenseRPS5 culture) orientation, as well as MEL cells transiently transfected with siRNAs specific for RPS5 gene silencing (MEL-RPS5siRNA culture) were assessed for their ability to fully execute their erythroid maturation program in culture. The data obtained thus far indicate that: a) MEL-antisenseRPS5 exhibit a pronounced delay in the initiation of differentiation, as well as an impairment of commitment, since the continuous presence of the inducer in culture is required for the cells to fully execute their erythroid maturation program. b) RNAi-mediating silencing of RPS5 gene expression resulted in the inability of MEL cells to differentiate; however, when these cells were allowed to recapitulate normal RPS5 gene expression levels they regained their differentiation capacity by accumulating high proportion of erythroid mature cells. c) Interestingly the latter, is accompanied by morphological changes of cells and an impairment of their proliferation and apoptosis potential. Such data for the first time correlate the RPS5 gene expression levels with the differentiation capacity of MEL cells in vitro, a fact that might also have implications in understanding ribosomopathies.

Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo.

BACKGROUND: Transcription elongation is frequently interrupted by pausing signals in DNA, with downstream effects on gene expression. Transcription errors also induce prolonged pausing, which can lead to a destabilized genome by interfering with DNA replication. Mechanisms of pausing associated with translocation blocks and misincorporation have been characterized in vitro, but not in vivo. RESULTS: We investigate the pausing pattern of RNA polymerase (RNAP) in Escherichia coli by a novel approach, combining native elongating transcript sequencing (NET-seq) with RNase footprinting of the transcripts (RNET-seq). We reveal that the G-dC base pair at the 5' end of the RNA-DNA hybrid interferes with RNAP translocation. The distance between the 5' G-dC base pair and the 3' end of RNA fluctuates over a three-nucleotide width. Thus, the G-dC base pair can induce pausing in post-translocated, pre-translocated, and backtracked states of RNAP. Additionally, a CpG sequence of the template DNA strand spanning the active site of RNAP inhibits elongation and induces G-to-A errors, which leads to backtracking of RNAP. Gre factors efficiently proofread the errors and rescue the backtracked complexes. We also find that pausing events are enriched in the 5' untranslated region and antisense transcription of mRNA genes and are reduced in rRNA genes. CONCLUSIONS: In E. coli, robust transcriptional pausing involves RNAP interaction with G-dC at the upstream end of the RNA-DNA hybrid, which interferes with translocation. CpG DNA sequences induce transcriptional pausing and G-to-A errors.

Antisense expression of the fasciclin-like arabinogalactan protein FLA6 gene in Populus inhibits expression of its homologous genes and alters stem biomechanics and cell wall composition in transgenic trees.

Fasciclin-like arabinogalactan proteins (FLAs) play important roles in the growth and development of roots, stems, and seeds in Arabidopsis. However, their biological functions in woody plants are largely unknown. In this work, we investigated the possible function of PtFLA6 in poplar. Quantitative real-time PCR, PtFLA6-yellow fluorescent protein (YFP) fusion protein subcellular localization, Western blotting, and immunohistochemical analyses demonstrated that the PtFLA6 gene was expressed specifically in the xylem of mature stem, and PtFLA6 protein was distributed ubiquitous in plant cells and accumulated predominantly in stem xylem fibres. Antisense expression of PtFLA6 in the aspen hybrid clone Poplar davidiana×Poplar bolleana reduced the transcripts of PtFLA6 and its homologous genes. Transgenic plants that showed a significant reduction in the transcripts of PtFLAs accumulated fewer PtFLA6 and arabinogalactan proteins than did the non-transgenic plants, leading to reduced stem flexural strength and stiffness. Further studies revealed that the altered stem biomechanics of transgenic plants could be attributed to the decreased cellulose and lignin composition in the xylem. In addition expression of some xylem-specific genes involved in cell wall biosynthesis was downregulated in these transgenic plants. All these results suggest that engineering the expression of PtFLA6 and its homologues could modulate stem mechanical properties by affecting cell wall composition in trees.

NanoFlares for the detection, isolation, and culture of live tumor cells from human blood.

Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in blood­so-called circulating tumor cells (CTCs)­may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable live-cell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy.

Dicer promotes transcription termination at sites of replication stress to maintain genome stability.

Nuclear RNAi is an important regulator of transcription and epigenetic modification, but the underlying mechanisms remain elusive. Using a genome-wide approach in the fission yeast S. pombe, we have found that Dcr1, but not other components of the canonical RNAi pathway, promotes the release of Pol II from the 3? end of highly transcribed genes, and, surprisingly, from antisense transcription of rRNA and tRNA genes, which are normally transcribed by Pol I and Pol III. These Dcr1-terminated loci correspond to sites of replication stress and DNA damage, likely resulting from transcription-replication collisions. At the rDNA loci, release of Pol II facilitates DNA replication and prevents homologous recombination, which would otherwise lead to loss of rDNA repeats especially during meiosis. Our results reveal a novel role for Dcr1-mediated transcription termination in genome maintenance and may account for widespread regulation of genome stability by nuclear RNAi in higher eukaryotes.

Different tobacco retrotransposons are specifically modulated by the elicitor cryptogein and reactive oxygen species.

Interactions of plant retrotransposons with different steps of biotic and abiotic stress-associated signaling cascades are still poorly understood. We perform here a finely tuned comparison of four tobacco retrotransposons (Tnt1, Tnt2, Queenti, and Tto1) responses to the plant elicitor cryptogein. We demonstrate that basal transcript levels in cell suspensions and plant leaves as well as the activation during the steps of defense signaling events are specific to each retrotransposon. Using antisense NtrbohD lines, we show that NtrbohD-dependent reactive oxygen species (ROS) production might act as negative regulator of retrotransposon activation.

In the sense of transcription regulation by G-quadruplexes: asymmetric effects in sense and antisense strands.

G-Quadruplexes occupy important regulatory regions in the genome. DNA G-quadruplexes in the promoter regions and RNA quadruplexes in the UTRs (untranslated regions) have been individually studied and variously implicated at different regulatory levels of gene expression. However, the formation of G-quadruplexes in the sense and antisense strands and their corresponding roles in gene regulation have not been studied in much detail. In the present study, we have elucidated the effect of strand asymmetry in this context. Using biophysical methods, we have demonstrated the formation of stable G-quadruplex structure in vitro using CD and UV melting. Additionally, ITC was employed to demonstrate that a previously reported selective G-quadruplex ligand was able to bind and stabilize the G-quadruplex in the present sequence. Further, we have shown using reporter constructs that although the DNA G-quadruplex in either strand can reduce translation efficiency, transcriptional regulation differs when G-quadruplex is present in the sense or antisense strand. We demonstrate that the G-quadruplex motif in the antisense strand substantially inhibits transcription, while when in the sense strand, it does not affect transcription, although it does ultimately reduce translation. Further, it is also shown that the G-quadruplex stabilizing ligand can enhance this asymmetric transcription regulation as a result of the increased stabilization of the G-quadruplex.

Knockdown of mitogen-activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos.

Arthropod-borne infectious diseases are responsible for nearly 1.5 million deaths annually across the globe, with malaria responsible for >50% of these deaths. Recent efforts to enhance malaria control have focused on developing genetically modified Anopheles mosquitoes that are resistant to malaria parasite infection by manipulating proteins that are essential to the immune response. Although this approach has shown promise, the lack of efficient genetic tools in the mosquito makes it difficult to investigate innate immunity using reverse genetics. Current gene knockdown strategies based on small interfering RNA are typically labourious, inefficient, and require extensive training. In the present study, we describe the use of morpholino antisense oligomers to knockdown MEK-ERK signalling in the midgut of Anopheles stephensi through a simple feeding protocol. Anti-MEK morpholino provided in a saline meal was readily ingested by female mosquitoes with minimal toxicity and resulted in knockdown of total MEK protein levels 3-4 days after morpholino feeding. Further, anti-MEK morpholino feeding attenuated inducible phosphorylation of the downstream kinase ERK and, as predicted by previous work, reduced parasite burden in mosquitoes infected with Plasmodium falciparum. To our knowledge, this is the first example of morpholino use for target protein knockdown via feeding in an insect vector. Our results suggest this method is not only efficient for studies of individual proteins, but also for studies of phenotypic control by complex cell signalling networks. As such, our protocol is an effective alternative to current methods for gene knockdown in arthropods.