siRNA Duplexes Research Articles

Impact of mobile and stationary phases on siRNA duplex stability in liquid chromatography

Nucleic acid duplexes are typically analyzed in non-denaturing conditions. Melting temperature (Tm) is the property used to measure duplex stability; however, it is not known how the chromatographic conditions and mobile phase composition affect the duplex stability. We employed differential scanning calorimetry (DSC) method to measure the melting temperature of chemically modified silencing RNA duplex (21 base pairs, 0.15 mM duplex concentration) in mobile phases commonly used in reversed-phase, ion-pair reversed-phase, size exclusion and hydrophilic interaction chromatography. We investigated mobile phases consisting of ammonium acetate, alkylammonium ion-pairing reagents, alkali-ion chlorides, magnesium chloride, and additives including methanol, ethanol, acetonitrile and hexafluoroisopropanol. Increasing buffer concentration enhanced the duplex stability (Tm was 67.1 – 78.2 °C for 10–100 mM [Na+] concentration). The melting temperature decreases with the increase in cation size (70.2 °C in 10 mM [Li+], 68.1 °C in 10 mM [NH4+], 65.6 °C in 10 mM [Cs+], and 56.6 °C in 10 mM [triethylammonium+] solutions). Inclusion of 20 % of organic solvent in buffer reduced the melting temperature by 1–3 °C, and denaturation power increases in the order MeOH<EtOH<MeCN. Next, we investigated the RNA duplex melting using selected chromatographic techniques and within the 10–90 °C column temperature range. Melting temperature obtained with size exclusion chromatography in 25 mM sodium phosphate buffer was ∼ 70 °C, about 5 °C lower compared to DSC values. The apparent melting temperature for a reversed-phase chromatography experiment was < 10 °C for 10 mM ammonium acetate mobile phase with acetonitrile eluent. Ion-pair reversed-phase with 10 mM triethylamine, 100 mM hexafluoroisopropanol and MeCN as eluent yielded Tm 49.3–54.9 °C. Hydrophilic interaction chromatography experiments with 10 mM ammonium acetate and ∼ 50 % acetonitrile mobile phase showed high duplex stability (Tm ∼ 80 °C). The observed chromatographic Tm values suggest that the formation of an organic/aqueous solvent layer on the chromatographic sorbent surface affects the duplex stability more significantly than the composition of the mobile phase alone.

2'-O-Alkyl-N 3-Methyluridine Functionalized Passenger Strand Improves RNAi Activity by Modulating the Thermal Stability.

Herein, we have demonstrated that the siRNA activity could be enhanced by incorporating the guide strand in the RISC complex through thermodynamic asymmetry caused by m3U-based destabilizing modifications. A nuclease stability study revealed that 2'-OMe-m3U and 2'-OEt-m3U modifications slightly improved the half-lives of siRNA strands in human serum. In the in vitro gene silencing assay, 2'-OMe-m3U modification at the 3'-overhang and cleavage site of the passenger strand in anti-renilla and anti-Bcl-2 siRNA duplexes were well-tolerated and exhibited improved gene silencing activity. However, gene silencing activity was attenuated when these modifications were incorporated at position 3 in the seed region of the antisense strand. The molecular modeling studies using these modifications at the seed region with the MID domain of hAGO2 explained that the 2'-alkoxy group makes steric interactions with the amino acid residues of the hAGO2 protein.

Key arginine residues in R2D2 dsRBD1 and dsRBD2 lead the siRNA recognition in Drosophila melanogaster RNAi pathway

In Drosophila melanogaster, Dcr-2:R2D2 heterodimer binds to the 21 nucleotide siRNA duplex to form the R2D2/Dcr-2 Initiator (RDI) complex, which is critical for the initiation of siRNA-induced silencing complex (RISC) assembly. During RDI complex formation, R2D2, a protein that contains three dsRNA binding domains (dsRBD), senses two aspects of the siRNA: thermodynamically more stable end (asymmetry sensing) and the 5′-phosphate (5'-P) recognition. Despite several detailed studies to date, the molecular determinants arising from R2D2 for performing these two tasks remain elusive. In this study, we have performed structural, biophysical, and biochemical characterization of R2D2 dsRBDs. We found that the solution NMR-derived structure of R2D2 dsRBD1 yielded a canonical α1-β1-β2-β3-α2 fold, wherein two arginine salt bridges provide additional stability to the R2D2 dsRBD1. Furthermore, we show that R2D2 dsRBD1 interacts with thermodynamically asymmetric siRNA duplex independent of its 5′-phosphorylation state, whereas R2D2 dsRBD2 prefers to interact with 5'-P siRNA duplex. The mutation of key arginine residues, R53 and R101, in concatenated dsRBDs of R2D2 results in a significant loss of siRNA duplex recognition. Our study deciphers the active roles of R2D2 dsRBDs by showing that dsRBD1 initiates siRNA recognition, whereas dsRBD2 senses 5′-phosphate as an authentic mark on functional siRNA.

Natural compound Byakangelicin suppresses breast tumor growth and motility by regulating SHP-1/JAK2/STAT3 signal pathway

Byakangelicin mostly obtained from the root of Angelica dahurica and has protective effect on liver injury and fibrosis. In addition, Byakangelicin, as a traditional medicine, is also used to treat colds, headache and toothache. Recent studies have shown that Byakangelicin exhibits anti-tumor function; however, the role of Byakangelicin in breast tumor progression and related mechanism has not yet been elucidated. Our study aims to investigate the role of Byakangelicin in breast tumor progression and the underlying mechanism. To measure the effect of Byakangelicin on JAK2/STAT3 signaling, a dual luciferase reporter assay and a Western blot assay were performed. CCK8, colony formation, apoptosis and cell invasion assays were used to examine the inhibitory potential of Byakangelicin on breast cancer cells. Additionally, SHP-1 was silenced by specific siRNA duplex and the function of SHP-1 on Byakangelicin-mediated inhibition of JAK2/STAT3 signaling was evaluated. Byakangelicin treatment significantly inhibited STAT3 transcriptional activity. In addition, Byakangelicin treatment blocked JAK2/STAT3 signaling in a dose-dependent manner. Byakangelicin-treated tumor cells showed a dramatically reduced proliferation, colony formation and invasion ability. Moreover, Byakangelicin remarkedly induced breast cancer cell apoptosis. Furthermore, Byakangelicin regulated the expression of SHP1.In conclusion, our current study indicated that Byakangelicin, a natural compound, inhibits SHP-1/JAK2/STAT3 signaling and thus blocks tumor growth and motility.

Intrathecal administration of a novel siRNA modality extends survival and improves motor function in the SOD1G93A ALS mouse model

Antisense oligonucleotides (ASOs) were the first modality to pioneer targeted gene knockdown in the treatment of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1). RNA interference (RNAi) is another mechanism of gene silencing in which short interfering RNAs (siRNAs) effectively degrade complementary transcripts. However, delivery to extrahepatic tissues like the CNS has been a bottleneck in the clinical development of RNAi. Herein, we identify potent siRNA duplexes for the knockdown of human SOD1 in which medicinal chemistry and conjugation to anaccessory oligonucleotide (ACO) enable activity in CNS tissues. Local delivery via intracerebroventricular or intrathecal injection into SOD1G93A mice delayed disease progression and extended animal survival with superior efficacy compared with an ASO resembling tofersen in sequence and chemistry. Treatment also prevented disease-related declines in motor function, including improvements in animal mobility, muscle strength, and coordination. The ACO itself does not target any specific complementary nucleic acid sequence; rather, it imparts benefits conducive to bioavailability and delivery through its chemistry. The complete conjugate (i.e., siRNA-ACO) represents a novel modality for delivery of duplex RNA (e.g., siRNA) to the CNS that is currently being tested in the clinic for treatment of ALS.

Induced Self-Assembly of Vitamin E-Spermine/siRNA Nanocomplexes via Spermine/Helix Groove-Specific Interaction for Efficient siRNA Delivery and Antitumor Therapy.

Gene therapy has been one of potential strategies for the treatment of different diseases, where efficient and safe gene delivery systems are also extremely in need. Current lipid nanoparticles (LNP) technology highly depends on the packing and condensation of nucleic acids with amine moieties. Here, an attempt to covalently link two natural compounds, spermine and vitamin E, is made to develop self-assembled nucleic acid delivery systems. Among them, the spermine moieties specifically interact with the major groove of siRNA helix through salt bridge interaction, while vitamin E moieties are located around siRNA duplex. Such amphiphilic vitamin E-spermine/siRNA complexes can further self-assemble into nanocomplexes like multiblade wheels. Further studies indicate that these siRNA nanocomplexes with the neutrally charged surface of vitamin E can enter cells via caveolin/lipid raft mediated endocytosis pathway and bypass lysosome trapping. With these self-assembled delivery systems, efficient siRNA delivery is successfully achieved for Eg5 and Survivin gene silencing as well as DNA plasmid delivery. Further in vivo study indicates that VE-Su-Sper/DSPE-PEG2000/siSurvivin self-assembled nanocomplexes can accumulate in cancer cells and gradually release siRNA in tumor tissues and show significant antitumor effect in vivo. The self-assembled delivery system provides a novel strategy for highly efficient siRNA delivery.

PCSK9 inhibition interrupts the cross-talk between keratinocytes and macrophages and prevents UVB-induced skin damage

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that promotes the degradation of low-density lipoprotein receptors. It is involved in hyperlipidemia as well as other diseases, such as cancer and skin inflammation. However, the detailed mechanism for PCSK9 on ultraviolet B (UVB)-induced skin lesions was not clear. Thus, the role and possible action mechanism of PCSK9 in UVB-induced skin damage in mice were studied here using siRNA and a small molecule inhibitor (SBC110736) against PCSK9. Immunohistochemical staining revealed a significant increase in PCSK9 expression after UVB exposure, indicating the possible role of PCSK9 in UVB damage. Skin damage, increase in epidermal thickness, and keratinocyte hyperproliferation were significantly alleviated after treatment with SBC110736 or siRNA duplexes, compared with that in the UVB model group. Notably, UVB exposure triggered DNA damage in keratinocytes, whereas substantial interferon regulatory factor 3 (IRF3) activation was observed in macrophages. Pharmacologic inhibition of STING or cGAS knockout significantly reduced UVB-induced damage. In the co-culture system, supernatant from UVB-treated keratinocyte induced IRF3 activation in macrophages. This activation was inhibited with SBC110736 and by PCSK9 knockdown. Collectively, our findings reveal that PCSK9 plays a critical role in the crosstalk between damaged keratinocytes and STING activation in macrophages. The interruption of this crosstalk by PCSK9 inhibition may be a potential therapeutic strategy for UVB-induced skin damage.

Biochemistry-informed design selects potent siRNAs against SARS-CoV-2

ABSTRACT RNA interference (RNAi) offers an efficient way to repress genes of interest, and it is widely used in research settings. Clinical applications emerged more recently, with 5 approved siRNAs (the RNA guides of the RNAi effector complex) against human diseases. The development of siRNAs against the SARS-CoV-2 virus could therefore provide the basis of novel COVID-19 treatments, while being easily adaptable to future variants or to other, unrelated viruses. Because the biochemistry of RNAi is very precisely described, it is now possible to design siRNAs with high predicted activity and specificity using only computational tools. While previous siRNA design algorithms tended to rely on simplistic strategies (raising fully complementary siRNAs against targets of interest), our approach uses the most up-to-date mechanistic description of RNAi to allow mismatches at tolerable positions and to force them at beneficial positions, while optimizing siRNA duplex asymmetry. Our pipeline proposes 8 siRNAs against SARS-CoV-2, and ex vivo assessment confirms the high antiviral activity of 6 out of 8 siRNAs, also achieving excellent variant coverage (with several 3-siRNA combinations recognizing each correctly-sequenced variant as of September 2022). Our approach is easily generalizable to other viruses as long as a variant genome database is available. With siRNA delivery procedures being currently improved, RNAi could therefore become an efficient and versatile antiviral therapeutic strategy.

Regulation of Sertoli cell function by planar cell polarity (PCP) protein Fjx1

Four-jointed box kinase 1 (Fjx1) is a planar cell polarity (PCP) protein and a member of the Fat (FAT atypical cadherin 1)/Dchs (Dachsous cadherin-related protein)/Fjx1 PCP complex. Fjx1 is also a non-receptor Ser/Thr protein kinase capable of phosphorylating Fat1 at is extracellular cadherin domains when it is being transported across the Golgi system. As such, Fjx1 is a Golgi-based regulator of Fat1 function by determining its extracellular deposition. Herein, Fjx1 was found to localize across the Sertoli cell cytoplasm, partially co-localized with the microtubules (MTs) across the seminiferous epithelium. It was most notable at the apical ES (ectoplasmic specialization) and basal ES, displaying distinctive stage-specific expression. The apical ES and basal ES are the corresponding testis-specific cell adhesion ultrastructures at the Sertoli-elongated spermatid interface and the Sertoli cell-cell interface, respectively, consistent with the role of Fjx1 as a Golgi-associated Ser/Thr kinase that modulates the Fat (and/or Dchs) integral membrane proteins. Its knockdown (KD) by RNAi using specific Fjx1 siRNA duplexes versus non-targeting negative control siRNA duplexes was found to perturb the Sertoli cell tight junction function, as well as perturbing the function and organization of MT and actin. While Fjx1 KD did not affect the steady-state levels of almost two dozens of BTB-associated Sertoli cell proteins, including structural and regulatory proteins, its KD was found to down-regulate Fat1 (but not Fat2, 3, and 4) and to up-regulate Dchs1 (but not Dchs2) expression. Based on results of biochemical analysis, Fjx1 KD was found to be capable of abolishing phosphorylation of its putative substrate Fat1 at its Ser/Thr sites, but not at its Tyr site, illustrating an intimate functional relationship of Fjx1 and Fat1 in Sertoli cells.

Terminal bridging of siRNA duplex at the ribose 2′ position controls strand bias and target sequence preference

Small interfering RNA (siRNA) and short hairpin RNA (shRNA) are widely used as RNA interference (RNAi) reagents. Recently, truncated shRNAs that trigger RNAi in a Dicer-independent manner have been developed. We generated a novel class of RNAi reagent, designated enforced strand bias (ESB) RNA, in which an siRNA duplex was chemically bridged between the 3' terminal overhang region of the guide strand and the 5' terminal nucleotide of the passenger strand. ESB RNA, which is chemically bridged at the 2' positions of ribose (2'-2' ESB RNA), functions in a Dicer-independent manner and was highly effective at triggering RNAi without the passenger strand-derived off-target effect. In addition, the 2'-2' ESB RNA exhibited a unique target sequence preference that differs from siRNA and silenced target sequences that could not be effectively suppressed by siRNA. Our results indicate that ESB RNA has the potential to be an effective RNAi reagent even when the target sequence is not suitable for siRNA.

SiRNAs to Knockdown Antiviral Chemokine-related Genes in FRhK-4 Cells

The objective of this study was to generate small interfering RNA (siRNA) to knockdown antiviral chemokine-related genes in fetal rhesus monkey kidney (FRhK-4) cells. We generated siRNA duplexes to suppress antiviral chemokines like CXCL10 and CCL4 in FRhK-4 cells by downregulating interferon regulatory factor (IRF) 3 and IRF7. Three siRNA duplexes (si-F-IRF3-1, si-F-IRF3-2, and si-F-IRF3-3) targeting IRF3, and one siRNA duplex (si-F-IRF7) targeting IRF7 were generated. A nontarget siRNA duplex was used as the negative control. The nontarget or target siRNA duplexes (si-F-IRF3-1, si-F-IRF3-2, si-F-IRF3-3, and si-F-IRF7) were transfected into FRhK-4 cells using transfection reagents, and they were then incubated at 37°C for 6 h with 5% CO2. After 6 h, the medium was removed, and fresh medium was added to each cell, and they were then incubated at 37°C for 48 h with 5% CO2. The transfected FRhK-4 cells were infected with hepatitis A virus (HAV) HM-175/18f (viral titer: 105 PFU/mL) and incubated at 37°C for 3 h with 5% CO2 for HAV propagation. The expression levels of chemokines, including CXCL10 and CCL4, under the regulation of IRF3 and IRF7 in the transfected FRhK-4 cells were measured using quantitative real-time polymerase chain reaction after 3 h of HAV infection. The results indicated that CXCL10 and CCL4 expression levels were decreased in FRhK-4 cells transfected with si-F-IRF3-1, si-F-IRF3-3, or si-F-IRF7 (p < 0.05) compared to those in the negative control. These results indicate that si-F-IRF3-1 and si-F-IRF3-3, and si-F-IRF7 successfully knocked down IRF3 and IRF7 in FRhK-4 cells, respectively and suppressed antiviral chemokines. These siRNAs could be used to suppress antiviral chemokines in FRhK-4 cells.

Analysis of siRNA with Denaturing and Non-Denaturing Ion-Pair Reversed-Phase Liquid Chromatography Methods

In this paper, we describe denaturing and non-denaturing ion-pair reversed-phase liquid chromatography (RPLC) methods for analyzing small interfering ribonucleic acid (siRNA). Drug formulations consisting of one or two siRNA duplexes were analyzed in non-denaturing conditions at a low column temperature to separate intact duplexes from single-stranded oligonucleotide contaminants and quantify them. In a denaturing method, we used an elevated column temperature to ensure the denaturation of siRNA duplexes into their single-stranded oligonucleotide counterparts. The goal of the denaturing LC method was to investigate the impurities in daughter oligonucleotides in siRNA. A column with chemically modified C18 column hardware showed improvements in analytical performance for nucleic acids compared to a conventional C18 stainless-steel column with the same pore size.

Broken up but still living together: how ARGONAUTE's retention of cleaved fragments explains its role during chromatin modification.

Throughout the eukaryotic kingdoms, small RNAs direct chromatin modification. ARGONAUTE proteins sit at the nexus of this process, linking the small RNA information to the programming of chromatin. ARGONAUTE proteins physically incorporate the small RNAs as guides to target specific regions of the genome. In this issue of Genes & Development, Wang and colleagues (pp. 103-118) add substantial new detail to the processes of ARGONAUTE RNA loading, preference, cleavage, and retention, which together accomplish RNA-directed chromatin modification. They show that after catalytic cleavage by the plant ARGONAUTE protein AGO4, the cleaved fragment remains bound. This happens during two distinct RNA cleavage reactions performed by AGO4: first for a passenger RNA strand of the siRNA duplex, and second for a nascent transcript at the target DNA locus. Cleaved fragment retention of the nascent transcript explains how the protein complex accumulates to high levels at the target locus, amplifying chromatin modification.

Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing, and sliced target retention.

RNA-directed DNA methylation in plants is guided by 24-nt siRNAs generated in parallel with 23-nt RNAs of unknown function. We show that 23-nt RNAs function as passenger strands during 24-nt siRNA incorporation into AGO4. The 23-nt RNAs are then sliced into 11- and 12-nt fragments, with 12-nt fragments remaining associated with AGO4. Slicing recapitulated with recombinant AGO4 and synthetic RNAs reveals that siRNAs of 21-24 nt, with any 5'-terminal nucleotide, can guide slicing, with sliced RNAs then retained by AGO4. In vivo, RdDM target locus RNAs that copurify with AGO4 also display a sequence signature of slicing. Comparing plants expressing slicing-competent versus slicing-defective AGO4 shows that slicing elevates cytosine methylation levels at virtually all RdDM loci. We propose that siRNA passenger strand elimination and AGO4 tethering to sliced target RNAs are distinct modes by which AGO4 slicing enhances RNA-directed DNA methylation.

A modular RNA delivery system comprising spherical nucleic acids built on endosome-escaping polymeric nanoparticles.

Nucleic acid therapeutics require delivery systems to reach their targets. Key challenges to be overcome include avoidance of accumulation in cells of the mononuclear phagocyte system and escape from the endosomal pathway. Spherical nucleic acids (SNAs), in which a gold nanoparticle supports a corona of oligonucleotides, are promising carriers for nucleic acids with valuable properties including nuclease resistance, sequence-specific loading and control of receptor-mediated endocytosis. However, SNAs accumulate in the endosomal pathway and are thus vulnerable to lysosomal degradation or recycling exocytosis. Here, an alternative SNA core based on diblock copolymer PMPC25-PDPA72 is investigated. This pH-sensitive polymer self-assembles into vesicles with an intrinsic ability to escape endosomes via osmotic shock triggered by acidification-induced disassembly. DNA oligos conjugated to PMPC25-PDPA72 molecules form vesicles, or polymersomes, with DNA coronae on luminal and external surfaces. Nucleic acid cargoes or nucleic acid-tagged targeting moieties can be attached by hybridization to the coronal DNA. These polymeric SNAs are used to deliver siRNA duplexes against C9orf72, a genetic target with therapeutic potential for amyotrophic lateral sclerosis, to motor neuron-like cells. By attaching a neuron-specific targeting peptide to the PSNA corona, effective knock-down is achieved at doses of 2 particles per cell.

Multifunctional magnetic nanocarriers for delivery of siRNA and shRNA plasmid to mammalian cells: Characterization, adsorption and release behaviors

Nucleic acids are promising candidates for treating various diseases. Nucleic acid is negatively charged and hydrophilic; therefore, it is not efficiently taken up by cells. Successful gene therapy requires the development of carriers for efficient delivery of gene-expressing DNA plasmid and small interfering RNA (siRNA) duplex. In this study, we developed MNP-CA-PEI, a citric acid (CA)-modified magnetic nanoparticle (MNP) cross-linked with polyethyleneimine (PEI), using carbonyldiimidazole as the crosslinker. The physical properties of MNP-CA-PEI (particle size, morphologies, surface coating, surface potentials, magnetic hystereses, superparamagnetic behaviors, and infrared spectra) were systematically characterized by transmission electron microscopy imaging, dynamic light scattering, thermogravimetric analysis, superconducting quantum interference device, and Fourier transform infrared spectroscopy. The adsorption isotherm and kinetics were determined by the Langmuir model, the Freundlich model, a pseudo-first-order equation, and a pseudo-second-order equation. MNP-CA-PEI could form polyelectrolyte complexes with negatively charged nucleic acids, enabling the efficient delivery of nucleic acids into cells. Using MNP-CA-PEI nanoparticles, we magnetically triggered the intracellular delivery of green fluorescence protein (GFP)-expressing DNA plasmid, plasmid-expressing short hairpin RNA (shRNA) against GFP, or siRNA targeting GFP into different cell lines. Nucleic acid/MNP-CA-PEI displayed the enhanced cellular uptake of GFP-expressing DNA plasmid, and it improved the silencing efficiency of shRNA and siRNA, determined by fluorescence imaging. Gene knockdowns mediated by shRNA and siRNA were also confirmed by a quantitative real-time polymerase chain reaction. MNP-CA-PEI delivered nucleic acids into cytosol through caveolae-mediated endocytosis. This study introduces a new MNP functionalization that can be used for the magnetically driven intracellular delivery of nucleic acids.

Synthesis, Derivatization and Photochemical Control of an ortho-Functionalized Tetrafluorinated Azobenzene-Modified siRNA.

We report the synthesis of an ortho-functionalized tetrafluorinated azobenzene phosphoramidite for its site-specific incorporation into RNA. The tetrafluorinated azobenzene is embedded within the antisense strand of an siRNA duplex to form an ortho-functionalized tetrafluorinated azobenzene-containing siRNA (F-siRNAzo). The F-siRNAzo is inactivated via trans to cis conversion with green light (530 nm), and reactivated with blue light (470 nm) via cis to trans conversion in cell culture. The long half-life and stability of the tetrafluorinated azobenzene unit allows for reversible control of the F-siRNAzo in cell culture for up 72 hours.

A model system for antiviral siRNA therapeutics using exosome-based delivery

Emerging viral diseases, such as Ebola, SARS, MERS, and the pathogen causing COVID-19, SARS-CoV-2, present a challenge for the development of therapeutics because of strict biosafety handling procedures and rapid mutation of their genomes. To facilitate the development of an adaptable and testable therapeutic model system, a colostrum exosome-based nanoparticle delivery system, EPM (exosome-PEI matrix), that overcomes stringent biosafety containment, was used to mimic the expression of viral proteins. This system would greatly expand the number of laboratories actively participating in the screening of potential therapeutics. EPM technology can deliver both plasmid DNA and siRNA to both simulate viral gene expression and screen potential antiviral siRNA therapeutics. Using this nanoplatform, three key SARS-CoV-2 proteins (the spike glycoprotein, nucleocapsid, and replicase) were expressed in vitro and in vivo. In vitro, several viral gene-targeting siRNAs were screened to determine knockdown efficiency, with some siRNA duplexes resulting in 80%–95% knockdown of corresponding protein expression. Moreover, in vivo experiments introducing the spike protein and nucleocapsid by EPM resulted in the production of antibodies against the viral antigen, measured up to 45 d after target delivery. Together, these findings support the efficacy of the EPM delivery system to establish a model for screening antiviral therapeutics-reduced biosafety level.

MARK2 and MARK4 Regulate Sertoli Cell BTB Dynamics Through Microtubule and Actin Cytoskeletons.

Microtubule affinity-regulating kinases (MARKs) are nonreceptor Ser/Thr protein kinases known to regulate cell polarity and microtubule dynamics in Caenorhabditis elegans, Drosophila, invertebrates, vertebrates, and mammals. An earlier study has shown that MARK4 is present at the ectoplasmic specialization and blood-testis barrier (BTB) in the seminiferous epithelium of adult rat testes. Here, we report the function of MARK4 and another isoform MARK2 in Sertoli cells at the BTB. Knockdown of MARK2, MARK4, or MARK2 and MARK4 by RNAi using the corresponding siRNA duplexes without apparent off-target effects was shown to impair tight junction (TJ)-permeability barrier at the Sertoli cell BTB. It also disrupted microtubule (MT)- and actin-based cytoskeletal organization within Sertoli cells. Although MARK2 and MARK4 were shown to share sequence homology, they likely regulated the Sertoli cell BTB and MT cytoskeleton differently. Disruption of the TJ-permeability barrier following knockdown of MARK4 was considerably more severe than loss of MARK2, though both perturbed the barrier. Similarly, loss of MARK2 affected MT organization in a different manner than the loss of MARK4. Knockdown of MARK2 caused MT bundles to be arranged around the cell periphery, whereas knockdown of MARK4 caused MTs to retract from the cell edge. These differences in effects on the TJ-permeability barrier are likely from the unique roles of MARK2 and MARK4 in regulating the MT cytoskeleton of the Sertoli cell.

Structure of the Dicer-2–R2D2 heterodimer bound to a small RNA duplex

In flies, Argonaute2 (Ago2) and small interfering RNA (siRNA) form an RNA-induced silencing complex to repress viral transcripts1. The RNase III enzyme Dicer-2 associates with its partner protein R2D2 and cleaves long double-stranded RNAs to produce 21-nucleotide siRNA duplexes, which are then loaded into Ago2 in a defined orientation2–5. Here we report cryo-electron microscopy structures of the Dicer-2–R2D2 and Dicer-2–R2D2–siRNA complexes. R2D2 interacts with the helicase domain and the central linker of Dicer-2 to inhibit the promiscuous processing of microRNA precursors by Dicer-2. Notably, our structure represents the strand-selection state in the siRNA-loading process, and reveals that R2D2 asymmetrically recognizes the end of the siRNA duplex with the higher base-pairing stability, and the other end is exposed to the solvent and is accessible by Ago2. Our findings explain how R2D2 senses the thermodynamic asymmetry of the siRNA and facilitates the siRNA loading into Ago2 in a defined orientation, thereby determining which strand of the siRNA duplex is used by Ago2 as the guide strand for target silencing.