siRNA Sequences Research Articles

Effects of silencing farnesyltransferase on the migration, invasion, and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells.

This study aimed to investigate the effects of farnesyltransferase (FTase) on the migration, invasion, and epithelial-mesenchymal transition (EMT) of SACC-LM and SACC-83 cells in salivary adenoid cystic carcinoma and determine the relative mechanism. Three small interfering RNA (siRNA) sequences were designed and constructed based on the human FTase gene sequence. The SACC-LM and SACC-83 cells in the logarithmic growth period were used, and the expression of FTase was suppressed by liposomal transient transfection. The tested cells were categorized as the FTase-siRNA-1, FTase-siRNA-2, and FTase-siRNA-3 groups. Both negative control group (NC-siRNA) and blank control group (only transfection reagent was added) were set. The mRNA expression of FTase and HRAS was detected by quantitive real-time polymerase chain reaction, and the silencing efficiency was determined. The expression levels of FTase, HRAS, protein kinase B (AKT), phospho-AKT, p65, phospho-p65 (Ser563), E-cadherin, vimentin, matrix metalloproteinase (MMP)-9 protein, and HRAS membrane protein were detected by Western blot. Transwell assay and wound healing assay were used to detect the invasion and migration abilities of cells. The relative expression of FTase mRNA and protein in the FTase-siRNA-1 group decreased compared with those in the control group (P<0.05). HRAS mRNA and total protein expression had no significant difference (P>0.05), and the relative expression of HRAS membrane protein decreased (P<0.05). The relative expression of E-cadherin increased (P<0.05), vimentin decreased (P<0.05), and MMP-9 decreased (P<0.05). There was no significant difference in the relative expression levels of the RAS/PI3K/AKT/nuclear factor-κB signaling pathway-related proteins AKT and p65 (P>0.05), but the relative expression levels of phospho-AKT and phospho-p65 decreased. The invasion and migration ability of the FTase-siRNA-1 group significantly decreased compared with that in the control group (P<0.05). Silencing FTase in vitro could effectively inhibit the invasion and migration of SACC-LM and SACC-83 cells by interfering with the localization of the HRAS membrane protein and regulating the RAS/PI3K/AKT/nuclear factor-κB signaling pathway to mediate EMT.

Analyzing siRNA Concentration, Complexation and Stability in Cationic Dendriplexes by Stem-Loop Reverse Transcription-qPCR.

RNA interference (RNAi) is a powerful therapeutic approach for messenger RNA (mRNA) level regulation in human cells. RNAi can be triggered by small interfering RNAs (siRNAs) which are delivered by non-viral carriers, e.g., dendriplexes. siRNA quantification inside carriers is essential in drug delivery system development. However, current siRNA measuring methods either are not very sensitive, only semi-quantitative or not specific towards intact target siRNA sequences. We present a novel reverse transcription real-time PCR (RT-qPCR)-based application for siRNA quantification in drug formulations. It enables specific and highly sensitive quantification of released, uncomplexed target siRNA and thus also indirect assessment of siRNA stability and concentration inside dendriplexes. We show that comparison with a dilution series allows for siRNA quantification, exclusively measuring intact target sequences. The limit of detection (LOD) was 4.2 pM (±0.2 pM) and the limit of quantification (LOQ) 77.8 pM (±13.4 pM) for uncomplexed siRNA. LOD and LOQ of dendriplex samples were 31.6 pM (±0 pM) and 44.4 pM (±9.0 pM), respectively. Unspecific non-target siRNA sequences did not decrease quantification accuracy when present in samples. As an example of use, we assessed siRNA complexation inside dendriplexes with varying nitrogen-to-phosphate ratios. Further, protection of siRNA inside dendriplexes from RNase A degradation was quantitatively compared to degradation of uncomplexed siRNA. This novel application for quantification of siRNA in drug delivery systems is an important tool for the development of new siRNA-based drugs and quality checks including drug stability measurements.

Abstract 4096: Development of novel anticancer RNA therapeutics with dual-targeting siRNA that silences ubiquitin family members

Abstract Ubiquitination is a metabolic process in eukaryotic cells that modifies proteins with ubiquitin leading to protein degradation via the ubiquitin-proteosome pathway. Cellular levels of ubiquitin are driven by the expression of RPS27A, UBA52, UBB and UBC. In cancer, the dysregulation of ubiquitination can affect various aspects of tumor progression including cell cycle regulation and gene expression. Ubiquitin B (UBB) gene has been shown to be over-expressed in some tumor types, including NSCLC and cervical cancer, and under-expressed in other tumors, in particularly a subset of ovarian cancers. A role for ubiquitin C (UBC) in cancer has not been well established but UBC has been described as a biomarker in renal cancer and CLL. In recent studies, repression of UBB in ovarian cancer cells was associated with sensitivity to UBC knockdown and demonstrated a synthetic lethal relationship. Since both UBB and UBC show substantial sequence homology, we explored the possibility of developing dual targeting siRNA that would silence both UBB and UBC. We identified unique individual siRNA sequences that efficiently silenced both UBB and UBC and led to rapid cytotoxicity in several cancer cell lines including the colon cancer cell line HCT-116, the breast cancer cell line SK-BR3, and the prostate cancer cell line 22Rv1. Treatment with either UBB-specific or UBC-specific siRNA did not show the same lethal phenotype. Dose response assays using the dual targeting siRNA designated U21 indicated EC50 for cytotoxicity between 50–500 pM in several cancer lines. Treatment of cancer cell lines with U21 leads to rapid apoptosis and cell death. Chemical modifications to pharmacologically stabilize the U21 siRNA sequence did not change its activity. The efficient cytotoxic activity of the U21 siRNA sequence has been integrated into our SeekR™ platform to create aptamer-siRNA chimeras for targeted delivery of the lethal U21 payload to specific cancer cell types. Citation Format: David O. Azorsa, David W. Lee, Marvin O'Ketch, Nathalie A. Azorsa, Ioanna Papacharalampous, Lizette A. Castaño, Jeffrey A. Kiefer, Spyro Mousses. Development of novel anticancer RNA therapeutics with dual-targeting siRNA that silences ubiquitin family members [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4096.

HOXC6 Regulates the Epithelial-Mesenchymal Transition through the TGF-β/Smad Signaling Pathway and Predicts a Poor Prognosis in Glioblastoma.

Background The HOX gene family of transcription factors, characterized by conserved homeodomains, is positively correlated with the resistance to chemotherapy drugs and poor prognosis, as well as the initiating potential of gliomas. However, there are few studies regarding the HOXC6 gene in glioma cells. Therefore, in the present study, we explored the regulatory roles and detailed mechanisms underlying the relationship between HOXC6 and the progression of GBM. Methods The expression levels and prognostic value of HOXC6 in GBM were evaluated using the data obtained from the GCCA, GEPIA, and ONCOMINE databases. The relationship between GBM prognosis and levels of HOXC6 was identified using Kaplan-Meier curves. The protein levels of HOXC6 in GBM and adjacent normal tissues were identified via Western blot and immunohistochemistry (IHC) staining methods. Lentiviruses containing full-length HOXC6 and HOXC6 specific siRNA sequences were used to overexpress and knock down, respectively, the expression of HOXC6 in U87 and U251 cells. The role of HOXC6 in the regulation of migration and proliferation of GBM cells was accessed using Transwell, wound healing, CCK-8, and colony formation assays. The activation of the TGF-β/Smad signaling pathway was detected via Western blotting. Results Compared to normal tissues and control cells, GBM tissues and cell lines showed higher expressions of HOXC6. The expression of HOXC6 was associated with disease-free and the overall survival of GBM patients. Additionally, positive correlations between the expression of HOXC6 and the migration and proliferation of GBM cells were observed in vitro. The mechanistic analyses indicated that HOXC6 exerts its promotive effect on the progression and invasion of glioma cells by promoting the activation of the EMT and TGF-β/Smad signaling pathways. Conclusions HOXC6 enhances the migration and proliferation of GBM by activating the EMT signaling pathway.

Surface-Functionalized Polymeric siRNA Nanoparticles for Tunable Targeting and Intracellular Delivery to Hematologic Cancer Cells.

To date, the application of RNA therapeutics to hematologic malignancies has been challenging owing to the resistance of blood cancer cells against conventional transfection methods. Herein, triple-targeting moiety-functionalized polymeric small interfering RNA (siRNA) nanoparticles were systematically developed for efficient targeted delivery of RNA therapeutics to hematologic cancer cells. Polymeric siRNAs were synthesized using rolling circle transcription and were surface-functionalized with three types of targeting moieties─a natural ligand and two additional combinations of cell-specific antibodies─for tunable targetability. As a proof of concept, the optimization of the hyaluronic acid/antibody conjugation ratio was performed for selective intracellular delivery to various non-Hodgkin's lymphoma (NHL) cell lines (Daudi, Raji, Ramos, and Toledo cells) via receptor-mediated endocytosis. The engineered nanoparticles showed almost 10-fold enhanced NHL-specific intracellular delivery and induced significant in vitro anticancer effects. This multitargeted nanoparticle platform may effectively support the intracellular delivery of polymeric siRNA sequences, and thus promote therapeutic effects in hematopoietic malignancies.

The role of siRNA in inhibition the biofilm formation as a first line of antibiotic resistance by regulation the MsrA drug efflux pump in Staphylococcus saprophyticus

The bacterium Staphylococcus saprophyticus (S. saprophyticus) is a common cause of urinary tract infections (UTI) in the community. To determine the effect of siRNA on the mRNA of the msrA gene in S. saprophyticus, 21-23 bp small interfering RNA (siRNA) duplexes were constructed against the mRNA of the msrA gene. The effect of siRNA on msrA mRNA expression was determined using reverse transcription PCR (RT-PCR). To assess changes in biofilm formation (BF) in response to siRNA activity, the usual tube technique was adopted. In vitro, msrA-siRNAs inhibited S. saprophyticus mRNA expression and activity. The efficacy of siRNA was determined by comparing the BF in S. saprophyticus before and after msrA-siRNA was introduced into the bacteria. In this investigation, two msrA-siRNA sequences, siRNA1 and siRNA2, were employed. qRT-PCR revealed that two msrA-siRNA sequences significantly suppressed the expression of msrA-mRNA, P = (0.010 and 0.002 ) respectively at (P&lt; 0.05) comparison with control. Regarding the BF results after treatment with two siRNA sequences, they were 5/6 (83.5%) and 4/6 (67%) negative formation and 1/6 (16.5%) and 2/6 (33%) positive formation, respectively compared with control.

Gene knockdown of HCN2 ion channels in the ventral tegmental area reduces ethanol consumption in alcohol preferring rats

ABSTRACT Background: Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) ionic channels are known to play a key role in the control of neuron excitability and have been proposed as a molecular target of ethanol. Previous studies in rats have shown that gene-induced overexpression of the HCN2 channel in the ventral tegmental area (VTA) increases the rewarding effects of ethanol and its intake by the animals. Objective: The aim of this work was to study the effects of VTA HCN2 gene knockdown in the voluntary ethanol consumption of alcohol-preferring UChB rats. Methods: Two lentiviral vectors were generated; LV-siRNA-HCN2, coding for a siRNA that elicited >95% reduction of HCN2 protein levels in vitro, and a control vector coding for a scrambled siRNA sequence. Female UChB naïve rats (n = 14) were microinjected into the VTA with LV-siRNA-HCN2 or the scrambled control vector (n = 11). Four days after, animals were given a daily free access to 10% ethanol and water for 10 days. Results: Rats treated with the LV-siRNA-HCN2 vector showed a ~ 70% reduction (p < .001) in their ethanol preference and ethanol intake compared to control animals. No changes were observed in the total fluid intake of both groups. HCN2 levels in the VTA were measured by Western blot showing a reduction of 40% (p < .05) in the rats injected with LV-siRNA-HCN2, compared to control animals. Conclusion: These results show that knockdown of HCN2 ionic channels in the VTA of UChB rats markedly reduces their voluntary ethanol intake, supporting the idea that HCN2 channels may constitute a therapeutic target for alcohol use disorders.

Knockdown of &lt;i&gt;FLT4&lt;/i&gt;, &lt;i&gt;Nup98&lt;/i&gt;, and &lt;i&gt;Nup205&lt;/i&gt; cellular genes as a suppressor for the viral activity of Influenza A/WSN/33 (H1N1) in A549 cell culture

Objectives. To evaluate the effect of cellular genes FLT4, Nup98, and Nup205 on the reproduction of the influenza A virus in A549 human lung cancer cell line.Methods. The work was carried out using the equipment of the center for collective use of the I.I. Mechnikov Research Institute of Vaccines and Sera (Russia). The virus-containing fluid was collected within three days from the moment of transfection and infection and the intensity of viral reproduction was assessed by viral titration and hemagglutination reaction. The viral RNA concentration was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR). To calculate statistically significant differences between groups, the nonparametric Mann–Whitney test was used.Results. In cells treated with small interfering RNAs (siRNAs) targeted at FLT4, Nup98, and Nup205 genes, a significant decrease in their expression and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) was observed at a multiplicity of infection (MOI) = 0.1. Additionally, it was found that a decrease in the expression of target genes using siRNA does not lead to a significant decrease in cell survival. The viral titer in cells treated with siRNA FLT4.2, Nup98.1, and Nup205 on the first day was lower by an average of 1.0 lg, and on the second and third days, by 2.2–2.3 lg, compared to cells treated with nonspecific siRNA. During real-time RT-PCR, a significant decrease in the concentration of viral RNA was observed with siRNA Nup98.1 (up to 190 times) and Nup205 (up to 30 times) on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained when determining the hemagglutinating activity of the virus. The hemagglutinating activity on the third day most strongly decreased in cells treated with siRNA Nup205 and FLT4.2 (16 times). In cells treated with siRNA FLT4.1, Nup98.1, and Nup98.2, hemagglutinating activity decreased by 8 times.Conclusions. In the present study, three cellular genes (FLT4, Nup98, and Nup205) were identified—the decrease in the expression of which effectively suppresses viral reproduction— and the original siRNA sequences were obtained. The results obtained are important for creating therapeutic and prophylactic medication, whose action is based on the RNA interference mechanism.

RNAi-mediated siRNA sequences to combat the COVID-19 pandemic with the inhibition of SARS-CoV2

The outbreak of the COVID-19 pandemic has cost five million lives to date, and was caused by a positive-sense RNA virus named SARS-CoV2. The lack of drugs specific to SARS-CoV2, leads us to search for an effective and specific therapeutic approach. Small interfering RNA (siRNA) is able to activate the RNA interference (RNAi) pathway to silence the specific targeted gene and inhibit the viral replication, and it has not yet attracted enough attention as a SARS-CoV2 antiviral agent. It could be a potential weapon to combat this pandemic until the completion of full scale, effective mass vaccination. For this study, specific siRNAs were designed using a web-based bioinformatics tool (siDirect2.0) against 14 target sequences. These might have a high probability of silencing the essential proteins of SARS-CoV2. such as: 3CLpro/Mpro/nsp5, nsp7, Rd-Rp/nsp12, ZD, NTPase/HEL or nsp13, PLpro/nsp3, envelope protein (E), spike glycoprotein (S), nucleocapsid phosphoprotein (N), membrane glycoprotein (M), ORF8, ORF3a, nsp2, and its respective 5′ and 3′-UTR. Among these potential drug targets, the majority of them contain highly conserved sequences; the rest are chosen on the basis of their role in viral replication and survival. The traditional vaccine development technology using SARS-CoV2 protein takes 6–8 months; meanwhile the virus undergoes several mutations in the candidate protein chosen for vaccine development. By the time the protein-based vaccine reaches the market, the virus would have undergone several mutations, such that the antibodies against the viral sequence may not be effective in restricting the newly mutated viruses. However, siRNA technology can make sequences based on real time viral mutation status. This has the potential for suppressing SARS-CoV2 viral replication, through RNAi technology.

Acetyltransferase P300 Inhibits the Proliferation, Invasion, and Migration of Esophageal Cancer via Survivin Acetylation

Background: Esophageal cancer is one of the primary death causes leading by cancer in the world, which is high morbidity and mortality. Epigenetic acetylation modification participates in and regulates the proliferation, invasion, and metastasis of various tumor cells, and the acetylation modification of tumor proteins involved by acetyltransferases may be one of the important mechanisms of esophageal carcinogenesis. The aim of this study was to investigate the correlation of acetyltransferase P300 and Survivin acetylation in esophageal cancer pathogenesis and its molecular mechanism. Methods: Fifty-five cases of esophageal cancer tissues and adjacent cancer tissues were collected, Survivin and P300 protein expression was measured by immunohistochemistry (SP) and protein blotting (Western Blot); Survivin acetylated protein levels were measured by coimmunoprecipitation (Co-IP); bioinformatics predicted the relationship between P300 and Survivin as the substrate, and fluorescence immunohistochemistry (IF) to verify the localization and expression of Survivin and P300 in esophageal cancer tissues; the correlation of Survivin acetylation, P300 and clinical cases characteristics was analyzed by statistics. P300 siRNA sequences were structured and transfected into EC109 cells. P300 protein expression and Survivin acetylated protein levels were determined by Co-IP. Cell viability was determined by the MTT assay, Scratch healing and Transwell chamber assay examined cell migration and invasion ability. Results: Survivin and P300 protein expression was significantly increased in human esophageal cancer tissues and EC109 cells. The Survivin protein was acetylated in esophageal cancer tissues and EC109 cells, and its protein acetylation rate was significantly increased; bioinformatics predicted that the acetyltransferase P300 could catalyze the acetylation of Survivin as a substrate, and the fluorescence immunohistochemistry confirmed that both Survivin and P300 simultaneously showed a high expression state in cancer tissues; Survivin acetylation and P300 expression; Survivin acetylation and P300 were closely related with esophageal cancer stage, tissue differentiation and lymph node metastasis. The vitro experiments showed that P300 RNA interference in esophageal cancer cells can significantly reduce the Survivin protein acetylation level, while inhibiting the survival, migration and invasion capacity of EC109 cells. Conclusion: P300 has a correlation with Survivin acetylation in the lespathological process of esophageal cancer, P300 may be an important upstream molecule of Survivin acetylation and has an important potential value in the diagnosis and treatment of esophageal cancer.

Tip60-siRNA Regulates ABCE1 Acetylation and Inhibits the Proliferation, Migration and Invasion of Esophageal Cancer via the Wnt Pathway

Objective: To investigate the effect of Tip60 gene silencing on the ABCE1 acetylation level and cell proliferation, migration and invasion in TE-1 cells of oesophageal cancer. Methods: The siRNA sequence of Tip60 was transfected with esophageal cancer TE-1 cells. Transfected siRNA vector cells were used as experimental group (si-T), siRNA no-loaded somatic cells were transfected as control group (si-NC), and untransfected TE-1 cells were used as blank group (Group N). ABCE1 mRNA was detected by qRT-PCR, the expression of ABCE1 protein, proliferation-related protein β catenin (β-catenin), GSK3β, and c-myc by Western blot, the protein acetylation level by immunoprecipitation, MTT assay for cell viability, scratch healing and Transwell compartment assay for migration and invasion ability. Results: After 48 h downregulation of the Tip60 gene, TE-1 cells showed no significant changes in the ABCE1 mRNA and protein expression. The acetylation level of ABCE1 decreased significantly, compared with the control group and the blank group. After Tip60 gene silencing, the expression of β-catenin and c-myc protein decreased, while the expression of GSK-3β protein increased. Cytofunctology experiments showed that the proliferative activity, migration and invasion ability of TE-1 cells in the experimental group were significantly inhibited. Conclusion: Down regulation of Tip60 gene can deacetylate ABCE1 protein and inhibit the proliferation activity, migration and invasion ability of esophageal cancer by blocking the conduction of Wnt signaling pathway.

An in-silico approach to design potential siRNAs against the ORF57 of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few human oncogenic viruses, which causes a variety of malignancies, including Kaposi's sarcoma, multicentric Castleman disease, and primary effusion lymphoma, particularly in human immunodeficiency virus patients. The currently available treatment options cannot always prevent the invasion and dissemination of this virus. In recent times, siRNA-based therapeutics are gaining prominence over conventional medications as siRNA can be designed to target almost any gene of interest. The ORF57 is a crucial regulatory protein for lytic gene expression of KSHV. Disruption of this gene translation will inevitably inhibit the replication of the virus in the host cell. Therefore, the ORF57 of KSHV could be a potential target for designing siRNA-based therapeutics. Considering both sequence preferences and target site accessibility, several online tools (i-SCORE Designer, Sfold web server) had been utilized to predict the siRNA guide strand against the ORF57. Subsequently, off-target filtration (BLAST), conservancy test (fuzznuc), and thermodynamics analysis (RNAcofold, RNAalifold, and RNA Structure web server) were also performed to select the most suitable siRNA sequences. Finally, two siRNAs were identified that passed all of the filtration phases and fulfilled the thermodynamic criteria. We hope that the siRNAs predicted in this study would be helpful for the development of new effective therapeutics against KSHV.

Caveolin-1 promotes cancer progression via inhibiting ferroptosis in head and neck squamous cell carcinoma.

BackgroundHead and neck squamous cell carcinoma (HNSCC) is an aggressive disease worldwide. Much progress has been made in exploring mechanisms and improving the therapy of HNSCC, but only a few studies have focused on the role of ferroptosis on HNSCC progression. The current study aimed to reveal the underlining mechanisms that caveolin‐1 (CAV1)‐ROS (reactive oxygen species)‐ferroptosis axis affect the process of HNSCC and discover novo therapeutic targets or strategies.MethodsThe role of CAV1 in ferroptosis was analyzed by FerrDb, and its clinical significance was examined by TCGA dataset of HNSCC. The expressions of caveolin‐1 (CAV1) in HNSCC tissues were measured by immunohistochemistry, western blot, and real‐time PCR assay. Three siRNA sequences were designed to silence CAV1 mRNA in HNSCC cells. Cell proliferation, colony formation, wound‐healing, and transwell assays were used to examine the proliferation, migration, and invasion of cancer cells. ROS evaluation and intracellular Fe2+ content assays were performed to examine the levels of ferroptosis.ResultsThrough the analysis with published data, CAV1 was found to overexpress in HNSCC than normal tissues, and was one of the vital suppressors of ferroptosis pathway. Our study showed that CAV1 was over expressed in HNSCC tissues and the high level of CAV1 predicted poorer prognosis. Further experiments indicated that CAV1 could inhibit the ferroptosis of cancer cells and promote the proliferation, migration and invasion.ConclusionsOverexpression of CAV1 in HNSCC inhibited the process of ferroptosis, leading to aggressive phenotypes, as well as worse prognosis. The regulatory pathway of CAV1 and ferroptosis are potential targets for designing diagnostic and combined therapeutic strategies for HNSCC patients.

Regulation effect of siRNA on β-globin in erythrocytes of hemoglobin H disease

目的研究靶向siRNA对体外定向分化培养的红系细胞β-珠蛋白的调控作用，为血红蛋白H（Hemoglobin H, HbH）病的基因治疗提供新的理论支持。方法①根据β-珠蛋白基因表达结果，在红系细胞中筛选出最佳siRNA序列及其有效作用剂量，检测有效剂量的最佳siRNA对红系细胞β-珠蛋白表达的调控和细胞凋亡的影响。②将有效剂量的最佳siRNA作用于HbH病红系细胞，检测转染后细胞β-珠蛋白表达、活性氧（ROS）和细胞凋亡率，综合评估转染siRNA对HbH病红系细胞的影响。结果①siRNA2可在转染后96 h内显著下调体外培养的红系细胞β-珠蛋白的表达，但对α-珠蛋白无明显调控作用。siRNA2的沉默效应和效应持续时间均与作用剂量有关。②siRNA2可下调HbH病红系细胞β-珠蛋白表达，减少细胞内ROS生成，并减少细胞凋亡率。结论靶向性siRNA可下调HbH病红系细胞β-珠蛋白表达，减少细胞内ROS产生，下调细胞凋亡率。

Maturation of siRNA by strand separation: Steered molecular dynamics study

RNA interference, particularly siRNA induced gene silencing is becoming an important avenue of modern therapeutics. The siRNA is delivered to the cells as short double helical RNA which becomes single stranded for forming the RISC complex. Significant experimental evidence is available for most of the steps except the process of the separation of the two strands. We have attempted to understand the pathway for double stranded siRNA (dsRNA) to single stranded (ssRNA) molecules using steered molecular dynamics simulations. As the process is completely unexplored we have applied force from all possible directions restraining all possible residues to convert dsRNA to ssRNA. We found pulling one strand along the helical axis direction restraining the far end of the other strand demands excessive force for ssRNA formation. Pulling a central residue of one strand, in a direction perpendicular to the helix axis, while keeping the base paired residue fixed requires intermediate force for strand separation. Moreover, we found that in this process the force requirement is quite high for the first bubble formation (nucleation energy) and the bubble propagation energies are quite small. We believe the success rate of the design of siRNA sequences for gene silencing may increase if this mechanistic knowledge is utilized for such a design process. Communicated by Ramaswamy H. Sarma

Next-generation sequencing identification and multiplex RT-PCR detection for viruses infecting cigar and flue-cured tobacco.

Early, precise and simultaneous identification of plant viruses is of great significance for preventing virus spread and reducing losses in agricultural yields. In this study, the identification of plant viruses from symptomatic samples collected from a cigar tobacco planting area in Deyang and a flue-cured tobacco planting area in Luzhou city, Sichuan Province, China, was conducted by deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform, and plant virus-specific contigs were generated based on virus-derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis were performed to determine the species or strains of these viruses. A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples, with a total of 105 contigs mapped to the closest plant viruses with lengths ranging from 34 ~ 1720nt. The results indicated that the major viruses were potato virus Y, Chilli veinal mottle virus, tobacco vein banding mosaic virus, tobacco mosaic virus and cucumber mosaic virus. Subsequently, a fast and sensitive multiplex reverse transcription polymerase chain reaction assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan. These results provide a theoretical basis and convenient methods for the rapid detection and control of viruses in cigar- and flue-cured tobacco.

Cucurbit chlorotic yellows virus infecting Rehmannia glutinosa was detected in China.

Rehmannia glutinosa Libosch. is a perennial herbaceous plant of the family Scrophulariaceae. Its roots can be used as traditional Chinese medicine. The asexual reproduction by vegetative organ of R. glutinosa lead to an increased viral disease that seriously affects its yield and quality (Kwak et al. 2020; Kwak et al. 2018; Ling and Liu 2009). Leaves of R. glutinosa in Wenxian County, Henan Province, China showed symptoms of chlorosis, mosaic and irregular yellow in August 2019. In general, the older leaves at the base or middle of the plant (sample 2# and 5#) first became irregular yellowing, followed by a gradual extend to the leaves at the top (Supplementary Fig. S1A). Six plants (2#, 3#, 5#, 7#, 8#, and 9#) with these symptoms were collected. The total RNA was extracted and its siRNAs were obtained. High-throughput siRNA sequencing (Sangon, Shanghai, China) was performed on Illumina Hiseq 2000 platform with paired-end method after siRNA library construction (NEBNext Ultra II RNA Library Prep Kit, NEB, UK). Sequencing files were treated with Illumina's CASAVA pipeline (version 1.8). The length of the resulting reads with adaptor removed were mostly distributed ranging from 21-24 nt (Supplementary Fig. S1B). The Velvet Software 0.7.31 (k=17) was taken to do de novo assembling, and the contigs (∼13,000, Contigs > 300 bp) were used to perform BLASTN against GenBank database. Two viruses, Rehmannia mosaic virus (ReMV) and cucurbit chlorotic yellows virus (CCYV), were frequently appeared in analyzed six symptomatic samples. To further identify the infection of CCYV to R. glutinosa, ten samples with virus-infected symptoms were randomly collected. Total protein and RNAs were extracted for RT-PCR and ELISA (HALING. Shanghai, China). A specific pair of primers (Supplementary Table S1) were designed to amplify the 753-bp length coat protein (CP) gene of CCYV. The result showed that two samples appeared a specific band of expected size on the agarose gel, which indicated that they were infected by CCYV (Supplementary Fig. S1C, Upper panel). The same result was obtained by ELISA assay (Supplementary Fig. S1D). The amplified CP fragment of CCYV was recycled and purified by TIANgel Midi Purification Kit (Tiangen, Beijing, China), followed by cloned into pMD19-T (TaKaRa, Dalian, China) and transformed into E. coli DH5a.Ten separate clones were selected and sequenced (Sangon, Shanghai, China) after PCR verification. The obtained sequences (GenBank accession No. MW521380 & MW521381) were analyzed by BLASTN and bioEdit software (version 7.2.3). The results showed 100% identity with the CCYV CP sequences that mainly derived from infected cucurbit. To confirm the occurrence and distribution of CCYV and ReMV in planting area, the other twenty-four samples (20 with chlorosis and stunt symptoms and 4 with invisible symptoms) were randomly collected for RT-PCR in different regions of Henan Province (Supplementary Table S1). The results showed that the CCYV and ReMV infection rate were 20.5% and 61.7%, respectively. Co-infection of the CCYV and ReMV was 5.8% in fields (Supplementary Table S2). In sum, these results indicated the CCYV can naturally infect R. glutinosa in China. CCYV is transmitted by white-fly in a semi-persistent manner and mainly damages cucurbits (Orfanidou et al. 2017). CCYV has been discovered in many places (Huang et al. 2010). To date, there is no report about CCYV infecting R. glutinosa in nature. This is the first report of CCYV naturally infect R. glutinosa in China.

AGO CLIP-based imputation of potent siRNA sequences targeting SARS-CoV-2 with antifibrotic miRNA-like activity

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with fatal pulmonary fibrosis. Small interfering RNAs (siRNAs) can be developed to induce RNA interference against SARS-CoV-2, and their susceptible target sites can be inferred by Argonaute crosslinking immunoprecipitation sequencing (AGO CLIP). Here, by reanalysing AGO CLIP data in RNA viruses, we delineated putative AGO binding in the conserved non-structural protein 12 (nsp12) region encoding RNA-dependent RNA polymerase (RdRP) in SARS-CoV-2. We utilised the inferred AGO binding to optimise the local RNA folding parameter to calculate target accessibility and predict all potent siRNA target sites in the SARS-CoV-2 genome, avoiding sequence variants. siRNAs loaded onto AGO also repressed seed (positions 2–8)-matched transcripts by acting as microRNAs (miRNAs). To utilise this, we further screened 13 potential siRNAs whose seed sequences were matched to known antifibrotic miRNAs and confirmed their miRNA-like activity. A miR-27-mimicking siRNA designed to target the nsp12 region (27/RdRP) was validated to silence a synthesised nsp12 RNA mimic in lung cell lines and function as an antifibrotic miR-27 in regulating target transcriptomes related to TGF-β signalling. siRNA sequences with an antifibrotic miRNA-like activity that could synergistically treat COVID-19 are available online (http://clip.korea.ac.kr/covid19).

COLLAGEN RELATED MUSCLE DISEASES: EP.62 Increasing allele selectivity of small interfering RNAs to target a dominant-negative glycine substitution causing a collagen VI-related dystrophy

Collagen VI-related dystrophies (COL6-RDs) are a group of frequently severe, congenital-onset muscular dystrophies for which there is no effective treatment. Dominant-negative mutations, in particular glycine substitutions and in-frame exon skips, are common in COL6A1, COL6A2 and COL6A3 genes, and they are capable of incorporating into the hierarchical assembly of the encoded collagen α1, α2 and α3 (VI) chains and as a consequence, produce a dysfunctional collagen VI extracellular matrix. RNA directed therapeutics offer great opportunities to silence dominant-negative mutations if designed to selectively target the mutant allele, as haploinsufficiency for the COL6 genes is not associated with clinical disease. To achieve the potential of RNA directed therapeutics for dominant conditions, the design of highly active, yet allele-specific, antisense oligonucleotides or siRNAs is required. Targeting single missense substitutions proves to be challenging, as the mutant target differs from the normal copy by only one nucleotide. To increase selectivity of siRNA silencing towards a common glycine substitution (G293R) in COL6A1, we deliberately introduced a second mismatch into the siRNA design, to destabilize hybridization to the normal allele while maintaining activity on the mutant transcript. A series of siRNAs in which the location of the second mismatch was located at various positions along the siRNA sequence were first screened in HEK293 cells with fluorescence reporters for both the normal and the mutant allele. We found at least two siRNAs that retain high silencing activity towards the mutant allele as evidenced by fluorescence and immunoblotting quantification, while preserving the expression of the normal allele. Treatment of patient-derived fibroblasts with these siRNAs effectively rescued COL6 matrix deposition. The design strategy that we tested here increases the differential targeting potential of specific mutations that were previously difficult to discriminate.

Co-Silencing of Tissue Transglutaminase-2 and Interleukin-15 Genes in a Celiac Disease Mimetic Mouse Model Using a Nanoparticle-in-Microsphere Oral System.

Celiac disease is a chronic inflammatory condition characterized by activation of the immune system in response to deamidation of gluten peptides brought about by tissue transglutaminase-2 (TG2). Overexpression of interleukin-15 (IL-15) in the intestinal epithelium and the lamina propria leads to the dysregulation of the immune system, leading to epithelial damage. The goal of this study was to develop an RNA interference therapeutic strategy for celiac disease using a combination of TG2 and IL-15 gene silencing in the inflamed intestine. TG2 and IL-15 silencing siRNA sequences, along with scrambled control, were encapsulated in a nanoparticle-in-microsphere oral system (NiMOS) and administered in a poly(I:C) mouse model of celiac disease. Single TG2 and IL-15 siRNA therapy and the combination showed effective gene silencing in vivo. Additionally, it was found that IL-15 gene silencing alone and combination in the NiMOS significantly reduced other proinflammatory cytokines. The tissue histopathology data also confirmed a reduction in immune cell infiltration and restoration of the mucosal architecture and barrier function in the intestine upon treatment. Overall, the results of this study show evidence that celiac disease can be potentially treated with an oral microsphere formulation using a combination of TG2 and IL-15 RNA interference therapeutic strategies.