Journal articles on the topic 'SiRNA'
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Rodriguez-Salazar, Carlos Andrés, Delia Piedad Recalde-Reyes, Juan Pablo Bedoya, Leonardo Padilla-Sanabria, Jhon Carlos Castaño-Osorio, and Maria Isabel Giraldo. "In Vitro Inhibition of Replication of Dengue Virus Serotypes 1–4 by siRNAs Bound to Non-Toxic Liposomes." Viruses 14, no. 2 (February 7, 2022): 339. http://dx.doi.org/10.3390/v14020339.
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Dengue virus is a ssRNA+ flavivirus, which produces the dengue disease in humans. Currently, no specific treatment exists. siRNAs regulate gene expression and have been used systematically to silence viral genomes; however, they require controlled release. Liposomes show favorable results encapsulating siRNA for gene silencing. The objective herein was to design and evaluate in vitro siRNAs bound to liposomes that inhibit DENV replication. siRNAs were designed against DENV1–4 from conserved regions using siDirect2.0 and Web-BLOCK-iT™ RNAiDesigner; the initial in vitro evaluation was carried out through transfection into HepG2 cells. siRNA with silencing capacity was encapsulated in liposomes composed of D-Lin-MC3-DMA, DSPC, Chol. Cytotoxicity, hemolysis, pro-inflammatory cytokine release and antiviral activity were evaluated using plaque assay and RT-qPCR. A working concentration of siRNA was established at 40 nM. siRNA1, siRNA2, siRNA3.1, and siRNA4 were encapsulated in liposomes, and their siRNA delivery through liposomes led to a statistically significant decrease in viral titers, yielded no cytotoxicity or hemolysis and did not stimulate release of pro-inflammatory cytokines. Finally, liposomes were designed with siRNA against DENV, which proved to be safe in vitro.
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Vijayaraghavan, Bhooma, Giri Padmanabhan, and Kumaresan Ramanathan. "Assessment of siRNA as a therapeutic molecule in Transient Receptor Potential Channel 5 gene silencing: a computational approach." Biomedical Research and Therapy 5, no. 1 (January 19, 2018): 1911–22. http://dx.doi.org/10.15419/bmrat.v5i1.405.
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Background: Ion channels play a crucial role in Glomerular filter damage that contributes to albuminuria. Transient receptor potential channel 5 (TRPC5) gene mediating such damage, demand for its target specific inhibition by RNA interference mechanism. Designing and selecting potential siRNA for TRPC5 gene silencing by computational analysis.
Materials & Methods: The mRNA sequence was retrieved from NCBI (National Center for Biotechnology Information). siRNA sequences were designed specifically from target genes using InvivoGen siRNA wizard software. Thermodynamic RNA-RNA interactions were used to evaluate the gene silencing efficiency by minimum free energy of hybridization; the hybridization structures were also obtained using BIBISERV2-RNAHybrid.
Results: The minimum free energy of hybridization of the three designed siRNAs (siRNA1, siRNA2 and siRNA3) were as follows: -28.2 kcal/mol, -24.1 kcal/mol, and-25.6 kcal/mol. Their corresponding GC content were 47.62%, 52.38% and 47.62%, respectively. Thus, siRNA1 had the least minimum free energy of hybridization (i.e. -28.2 kcal/mol) with low GC content (47.62%), and high linearity with minimal h-b index and loop structure.
Conclusion: RNAi therapy can provide a new platform for efficient and targeted therapeutics. Further in vivo investigations are necessary to further validate their efficacy.
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Yu, Ji-wei, Shou-lian Wang, Ju-gang Wu, Rui-qi Lu, Xiao-chun Ni, Cheng Cai, and Bo-jian Jiang. "Study on the Biological Characteristics of CD133+ Cells Interfered by RNA Interference in Gastric Cancer." ISRN Gastroenterology 2014 (March 19, 2014): 1–11. http://dx.doi.org/10.1155/2014/329519.
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Background. To detect the changes of biological characteristics in gastric cancer cells interfered by CD133-specific small interfering RNA (siRNA). Methods. First to select the siRNA which has the strongest interference effect among 3 siRNAs (i.e., siRNA1, siRNA2, and siRNA3) in KATO-III cells by RT-PCR and Western blotting assays. Then, CD133+ cells were sorted out from KATO-III cells using an immunomagnetic bead sorting method and transfected with the selected siRNA. Furthermore, the proliferating characteristics, the antichemotherapeutic assessment, Transwell invasion assay, monoclonal sphere formation assay, and subcutaneous transplanted tumor formation assay in nude mice were investigated. Results. siRNA3 showed the strongest interference effect in KATO-III cells. As compared to the uninterfered control group, the CD133+ cells treated by siRNA3 showed significant decreases in the abilities of proliferation, invasion, clone sphere formation, and resistance to antitumour drugs as well as the weight and size of the transplanted tumor, which was nearly similar to that of CD133− cells. Additionally, the protein expression level of the EMT factor E-cadherin increased while those of EMT-related Snail and N-cadherin decreased in CD133+ cells interfered by siRNA3. Conclusion. Inhibition of CD133 gene expression reduces the abilities of gastric cancer cells in proliferation, invasion, clonal sphere formation, and chemoresistance as well as tumor formation in nude mice.
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Kadhim, Kadhim Kamil, Anas Y. Al-Hayawi, and Fawzyia A. R. Ibrahim. "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." International journal of health sciences 6, S1 (March 20, 2022): 1336–44. http://dx.doi.org/10.53730/ijhs.v6ns1.4904.
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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< 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.
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Shmushkovich, Taisia, Kathryn R. Monopoli, Diana Homsy, Dmitriy Leyfer, Monica Betancur-Boissel, Anastasia Khvorova, and Alexey D. Wolfson. "Functional features defining the efficacy of cholesterol-conjugated, self-deliverable, chemically modified siRNAs." Nucleic Acids Research 46, no. 20 (August 29, 2018): 10905–16. http://dx.doi.org/10.1093/nar/gky745.
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Abstract Progress in oligonucleotide chemistry has produced a shift in the nature of siRNA used, from formulated, minimally modified siRNAs, to unformulated, heavily modified siRNA conjugates. The introduction of extensive chemical modifications is essential for conjugate-mediated delivery. Modifications have a significant impact on siRNA efficacy through interference with recognition and processing by RNAi enzymatic machinery, severely restricting the sequence space available for siRNA design. Many algorithms available publicly can successfully predict the activity of non-modified siRNAs, but the efficiency of the algorithms for designing heavily modified siRNAs has never been systematically evaluated experimentally. Here we screened 356 cholesterol-conjugated siRNAs with extensive modifications and developed a linear regression-based algorithm that effectively predicts siRNA activity using two independent datasets. We further demonstrate that predictive determinants for modified and non-modified siRNAs differ substantially. The algorithm developed from the non-modified siRNAs dataset has no predictive power for modified siRNAs and vice versa. In the context of heavily modified siRNAs, the introduction of chemical asymmetry fully eliminates the requirement for thermodynamic bias, the major determinant for non-modified siRNA efficacy. Finally, we demonstrate that in addition to the sequence of the target site, the accessibility of the neighboring 3′ region significantly contributes to siRNA efficacy.
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Guo, Yong, Hongyan Guo, Liang Zhang, Hongying Xie, Xin Zhao, Fangxun Wang, Ze Li, et al. "Genomic Analysis of Anti-Hepatitis B Virus (HBV) Activity by Small Interfering RNA and Lamivudine in Stable HBV-Producing Cells." Journal of Virology 79, no. 22 (November 15, 2005): 14392–403. http://dx.doi.org/10.1128/jvi.79.22.14392-14403.2005.
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ABSTRACT Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Small interfering RNA (siRNA) and lamivudine have been shown to have anti-HBV effects through different mechanisms. However, assessment of the genome-wide effects of siRNA and lamivudine on HBV-producing cell lines has not been reported, which may provide a clue to interrogate the HBV-cell interaction and to evaluate the siRNA's side effect as a potential drug. In the present study, we designed seven siRNAs based on the conserved HBV sequences and tested their effects on the expression of HBV genes following sorting of siRNA-positive cells. Among these seven siRNAs, siRNA-1 and siRNA-7 were found to effectively suppress HBV gene expression. We further addressed the global gene expression changes in stable HBV-producing cells induced by siRNA-1 and siRNA-7 by use of human genome-wide oligonucleotide microarrays. Data from the gene expression profiling indicated that siRNA-1 and siRNA-7 altered the expression of 54 and 499 genes, respectively, in HepG2.2.15 cells, which revealed that different siRNAs had various patterns of gene expression profiles and suggested a complicated influence of siRNAs on host cells. We further observed that 18 of these genes were suppressed by both siRNA-1 and siRNA-7. Interestingly, seven of these genes were originally activated by HBV, which suggested that these seven genes might be involved in the HBV-host cell interaction. Finally, we have compared the effects of siRNA and lamivudine on HBV and host cells, which revealed that siRNA is more effective at inhibiting HBV expression at the mRNA and protein level in vitro, and the gene expression profile of HepG2.2.15 cells treated by lamivudine is totally different from that seen with siRNA.
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Bolhassani, Azam, and Alireza Milani. "Small Interfering RNAs and their Delivery Systems: A Novel Powerful Tool for the Potential Treatment of HIV Infections." Current Molecular Pharmacology 13, no. 3 (July 9, 2020): 173–81. http://dx.doi.org/10.2174/1874467212666191023120954.
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: Small interfering RNAs (siRNAs) have rapidly developed into biomedical research as a novel tool for the potential treatment of various human diseases. They are based on altered gene expression. In spite of the availability of highly active antiretroviral therapy (HAART), there is a specific interest in developing siRNAs as a therapeutic agent for human immunodeficiency virus (HIV) due to several problems including toxicity and drug resistance along with long term treatment. The successful use of siRNAs for therapeutic goals needs safe and effective delivery to specific cells and tissues. Indeed, the efficiency of gene silencing depends on the potency of the carrier used for siRNA delivery. The combination of siRNA and nano-carriers is a potent method to prevent the limitations of siRNA formulation. Three steps were involved in non-viral siRNA carriers such as the complex formation of siRNA with a cationic carrier, conjugation of siRNA with small molecules, and encapsulation of siRNA within nanoparticles. : In this mini-review, the designed siRNAs and their carriers are described against HIV-1 infections both in vitro and in vivo.
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Wang, Xuan, and Fa Zhang. "Prediction of siRNA Efficacy Using BP Neural Network and Support Vector Machine." Applied Mechanics and Materials 701-702 (December 2014): 214–18. http://dx.doi.org/10.4028/www.scientific.net/amm.701-702.214.
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RNA interference (RNAi) is a mechanism for sequence-specific, post-transcriptional down-regulation of gene expression. The success of RNAi gene silencing depends on siRNA feature design. The shortcoming of previously reported methods which design siRNA sequences based on limited rules is that they are difficult to accurately predict the efficacy that a candidate siRNA sequence will silence the target gene. With validated siRNA databases have been developed in recent years, machine learning methods can be applied to predict siRNA accuracy and optimize design. This paper proposed a combined prediction method of BP neural network and support vector machine (SVM) for selecting effective siRNA sequences. With SVM, siRNA sequences were classified into effective or ineffective siRNAs. Subsequently, BP neural network model with great learning ability selected highly effective candidate sequences from effective siRNAs. We applied this method to published siRNAs datasets, and the experimental results confirmed good prediction capability.
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Chernikov, Ivan V., Ulyana A. Ponomareva, and Elena L. Chernolovskaya. "Structural Modifications of siRNA Improve Its Performance In Vivo." International Journal of Molecular Sciences 24, no. 2 (January 4, 2023): 956. http://dx.doi.org/10.3390/ijms24020956.
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The use of small interfering RNA (siRNA) in the clinic gives a wide range of possibilities for the treatment of previously incurable diseases. However, the main limitation for biomedical applications is their delivery to target cells and organs. Currently, delivery of siRNA to liver cells is a solved problem due to the bioconjugation of siRNA with N-acetylgalactosamine; other organs remain challenging for siRNA delivery to them. Despite the important role of the ligand in the composition of the bioconjugate, the structure and molecular weight of siRNA also play an important role in the delivery of siRNA. The basic principle is that siRNAs with smaller molecular weights are more efficient at entering cells, whereas siRNAs with larger molecular weights have advantages at the organism level. Here we review the relationships between siRNA structure and its biodistribution and activity to find new strategies for improving siRNA performance.
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Rajeswaran, Rajendran, Victor Golyaev, Jonathan Seguin, Anna S. Zvereva, Laurent Farinelli, and Mikhail M. Pooggin. "Interactions of Rice Tungro Bacilliform Pararetrovirus and Its Protein P4 with Plant RNA-Silencing Machinery." Molecular Plant-Microbe Interactions® 27, no. 12 (December 2014): 1370–78. http://dx.doi.org/10.1094/mpmi-07-14-0201-r.
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Small interfering RNA (siRNA)-directed gene silencing plays a major role in antiviral defense. Virus-derived siRNAs inhibit viral replication in infected cells and potentially move to neighboring cells, immunizing them from incoming virus. Viruses have evolved various ways to evade and suppress siRNA production or action. Here, we show that 21-, 22-, and 24-nucleotide (nt) viral siRNAs together constitute up to 19% of total small RNA population of Oryza sativa plants infected with Rice tungro bacilliform virus (RTBV) and cover both strands of the RTBV DNA genome. However, viral siRNA hotspots are restricted to a short noncoding region between transcription and reverse-transcription start sites. This region generates double-stranded RNA (dsRNA) precursors of siRNAs and, in pregenomic RNA, forms a stable secondary structure likely inaccessible to siRNA-directed cleavage. In transient assays, RTBV protein P4 suppressed cell-to-cell spread of silencing but enhanced cell-autonomous silencing, which correlated with reduced 21-nt siRNA levels and increased 22-nt siRNA levels. Our findings imply that RTBV generates decoy dsRNA that restricts siRNA production to the structured noncoding region and thereby protects other regions of the viral genome from repressive action of siRNAs, while the viral protein P4 interferes with cell-to-cell spread of antiviral silencing.
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Han, Ye, Yuanning Liu, Hao Zhang, Fei He, Chonghe Shu, and Liyan Dong. "Utilizing Selected Di- and Trinucleotides of siRNA to Predict RNAi Activity." Computational and Mathematical Methods in Medicine 2017 (2017): 1–12. http://dx.doi.org/10.1155/2017/5043984.
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Small interfering RNAs (siRNAs) induce posttranscriptional gene silencing in various organisms. siRNAs targeted to different positions of the same gene show different effectiveness; hence, predicting siRNA activity is a crucial step. In this paper, we developed and evaluated a powerful tool named “siRNApred” with a new mixed feature set to predict siRNA activity. To improve the prediction accuracy, we proposed 2-3NTs as our new features. A Random Forest siRNA activity prediction model was constructed using the feature set selected by our proposed Binary Search Feature Selection (BSFS) algorithm. Experimental data demonstrated that the binding site of the Argonaute protein correlates with siRNA activity. “siRNApred” is effective for selecting active siRNAs, and the prediction results demonstrate that our method can outperform other current siRNA activity prediction methods in terms of prediction accuracy.
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de Brito e Cunha, Danielle, Ana Beatriz Teixeira Frederico, Tamiris Azamor, Juliana Gil Melgaço, Patricia Cristina da Costa Neves, Ana Paula Dinis Ano Bom, Tatiana Martins Tilli, and Sotiris Missailidis. "Biotechnological Evolution of siRNA Molecules: From Bench Tool to the Refined Drug." Pharmaceuticals 15, no. 5 (May 5, 2022): 575. http://dx.doi.org/10.3390/ph15050575.
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The depth and versatility of siRNA technologies enable their use in disease targets that are undruggable by small molecules or that seek to achieve a refined turn-off of the genes for any therapeutic area. Major extracellular barriers are enzymatic degradation of siRNAs by serum endonucleases and RNAases, renal clearance of the siRNA delivery system, the impermeability of biological membranes for siRNA, activation of the immune system, plasma protein sequestration, and capillary endothelium crossing. To overcome the intrinsic difficulties of the use of siRNA molecules, therapeutic applications require nanometric delivery carriers aiming to protect double-strands and deliver molecules to target cells. This review discusses the history of siRNAs, siRNA design, and delivery strategies, with a focus on progress made regarding siRNA molecules in clinical trials and how siRNA has become a valuable asset for biopharmaceutical companies.
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Tuschl, Thomas. "Annealing siRNAs to Produce siRNA Duplexes." Cold Spring Harbor Protocols 2006, no. 1 (January 1, 2006): pdb.prot4340. http://dx.doi.org/10.1101/pdb.prot4340.
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Yoshikawa, Manabu, Yong-Woon Han, Hirofumi Fujii, Shu Aizawa, Tatsuya Nishino, and Masayuki Ishikawa. "Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis." Proceedings of the National Academy of Sciences 118, no. 34 (August 18, 2021): e2102885118. http://dx.doi.org/10.1073/pnas.2102885118.
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Small interfering RNAs (siRNAs) are often amplified from transcripts cleaved by RNA-induced silencing complexes (RISCs) containing a small RNA (sRNA) and an Argonaute protein. Amplified siRNAs, termed secondary siRNAs, are important for reinforcement of target repression. In plants, target cleavage by RISCs containing 22-nucleotide (nt) sRNA and Argonaute 1 (AGO1) triggers siRNA amplification. In this pathway, the cleavage fragment is converted into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase 6 (RDR6), and the dsRNA is processed into siRNAs by Dicer-like proteins. Because nonspecific RDR6 recruitment causes nontarget siRNA production, it is critical that RDR6 is specifically recruited to the target RNA that serves as a template for dsRNA formation. Previous studies showed that Suppressor of Gene Silencing 3 (SGS3) binds and stabilizes 22-nt sRNA–containing AGO1 RISCs associated with cleaved target, but how RDR6 is recruited to targets cleaved by 22-nt sRNA–containing AGO1 RISCs remains unknown. Here, using cell-free extracts prepared from suspension-cultured Arabidopsis thaliana cells, we established an in vitro system for secondary siRNA production in which 22-nt siRNA–containing AGO1-RISCs but not 21-nt siRNA–containing AGO1-RISCs induce secondary siRNA production. In this system, addition of recombinant Silencing Defective 5 (SDE5) protein remarkably enhances secondary siRNA production. We show that RDR6 is recruited to a cleavage fragment by 22-nt siRNA–containing AGO1-RISCs in coordination with SGS3 and SDE5. The SGS3–SDE5–RDR6 multicomponent recognition system and the poly(A) tail inhibition may contribute to securing specificity of siRNA amplification.
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Sijen, Titia, Florian A. Steiner, Karen L. Thijssen, and Ronald H. A. Plasterk. "Secondary siRNAs Result from Unprimed RNA Synthesis and Form a Distinct Class." Science 315, no. 5809 (December 7, 2006): 244–47. http://dx.doi.org/10.1126/science.1136699.
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In Caenorhabditis elegans, an effective RNA interference (RNAi) response requires the production of secondary short interfering RNAs (siRNAs) by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22-nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non–RISC (RNA-induced silencing complex)–cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5′ di- or triphosphates, and are only in the minority associated with RDE-1, the RNAi-specific Argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product.
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Song, Erwei, Sang-Kyung Lee, Derek M. Dykxhoorn, Carl Novina, Dong Zhang, Keith Crawford, Jan Cerny, et al. "Sustained Small Interfering RNA-Mediated HumanImmunodeficiency Virus Type 1 Inhibition in PrimaryMacrophages." Journal of Virology 77, no. 13 (July 1, 2003): 7174–81. http://dx.doi.org/10.1128/jvi.77.13.7174-7181.2003.
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ABSTRACT Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.
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Dong, Xialan, and Weifan Zheng. "Cheminformatics Modeling of Gene Silencing for Both Natural and Chemically Modified siRNAs." Molecules 27, no. 19 (September 28, 2022): 6412. http://dx.doi.org/10.3390/molecules27196412.
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In designing effective siRNAs for a specific mRNA target, it is critically important to have predictive models for the potency of siRNAs. None of the published methods characterized the chemical structures of individual nucleotides constituting a siRNA molecule; therefore, they cannot predict the potency of gene silencing by chemically modified siRNAs (cm-siRNA). We propose a new approach that can predict the potency of gene silencing by cm-siRNAs, which characterizes each nucleotide (NT) using 12 BCUT cheminformatics descriptors describing its charge distribution, hydrophobic and polar properties. Thus, a 21-NT siRNA molecule is described by 252 descriptors resulting from concatenating all the BCUT values of its composing nucleotides. Partial Least Square is employed to develop statistical models. The Huesken data (2431 natural siRNA molecules) were used to perform model building and evaluation for natural siRNAs. Our results were comparable with or superior to those from Huesken’s algorithm. The Bramsen dataset (48 cm-siRNAs) was used to build and test the models for cm-siRNAs. The predictive r2 of the resulting models reached 0.65 (or Pearson r values of 0.82). Thus, this new method can be used to successfully model gene silencing potency by both natural and chemically modified siRNA molecules.
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Marinho, H. Susana, Paulo Marcelino, Helena Soares, and Maria Luísa Corvo. "Gene Silencing using siRNA for Preventing Liver Ischaemia-Reperfusion Injury." Current Pharmaceutical Design 24, no. 23 (October 24, 2018): 2692–700. http://dx.doi.org/10.2174/1381612824666180807124356.
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Background: Ischaemia-reperfusion injury (IRI), a major complication occurring during organ transplantation, involves an initial ischemia insult, due to loss of blood supply, followed by an inflammation-mediated reperfusion injury. A variety of molecular targets and pathways involved in liver IRI have been identified. Gene silencing through RNA interference (RNAi) by means of small interference RNA (siRNA) targeting mediators of IRI is a promising therapeutic approach. Objective: This study aims at reviewing the use of siRNAs as therapeutic agents to prevent IRI during liver transplantation. Method: We review the crucial choice of siRNA targets and the advantages and problems of the use of siRNAs. Results: We propose possible targets for siRNA therapy during liver IRI. Moreover, we discuss how drug delivery systems, namely liposomes, may improve siRNA therapy by increasing siRNA stability in vivo and avoiding siRNA off-target effects. Conclusion: siRNA therapeutic potential to preclude liver IRI can be improved by a better knowledge of what molecules to target and by using more efficient delivery strategies.
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Epanchintseva, Anna V., Julia E. Poletaeva, Anton S. Dome, Ilya S. Dovydenko, Inna A. Pyshnaya, and Elena I. Ryabchikova. "Chemical Modifications Influence the Number of siRNA Molecules Adsorbed on Gold Nanoparticles and the Efficiency of Downregulation of a Target Protein." Nanomaterials 12, no. 24 (December 14, 2022): 4450. http://dx.doi.org/10.3390/nano12244450.
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Small interfering RNAs (siRNAs) are a powerful tool for specific suppression of protein synthesis in the cell, and this determines the attractiveness of siRNAs as a drug. Low resistance of siRNA to nucleases and inability to enter into target cells are the most crucial issues in developing siRNA-based therapy. To face this challenge, we designed multilayer nanoconstruct (MLNC) with AuNP core bearing chemically modified siRNAs. We applied chemical modifications 2′-OMe and 2′-F substitutions as well as their combinations with phosphoryl guanidine group in the internucleotide phosphate. The effect of modification on the efficiency of siRNA loading into nanocarriers was examined. The introduction of the internucleotide modifications into at least one of the strands raised the efficiency of siRNA adsorption on the surface of gold core. We also tested the stability of modified siRNA adsorbed on gold core in the presence of serum. Based on loading efficiency and stability, MLNCs with the most siRNA effective cargo were selected, and they showed an increase in biological activity compared to control MLNCs. Our study demonstrated the effect of chemical modifications of siRNA on its binding to the AuNP-based carrier, which directly affects the efficiency of target protein expression inhibition.
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Li, Chenxin, Jonathan I. Gent, Hengping Xu, Hong Fu, Scott D. Russell, and Venkatesan Sundaresan. "Resetting of the 24-nt siRNA landscape in rice zygotes." Genome Research 32, no. 2 (December 23, 2021): 309–23. http://dx.doi.org/10.1101/gr.275981.121.
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The zygote, a totipotent stem cell, is crucial to the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells—the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Owing to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Because siRNAs serve as both regulators and outputs of the epigenome, we characterized small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibit extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation is accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ∼70% of the zygote siRNA loci do not overlap any egg cell siRNA loci. Newly detected siRNA loci in zygote are gene-proximal and not associated with centromeric heterochromatin, similar to canonical siRNAs, in sharp contrast to gametic siRNA loci that are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci are associated with high DNA methylation in the mature embryo. Thus, the zygote begins transitioning before the first embryonic division to an siRNA profile that is associated with future RdDM in embryogenesis. These findings indicate that, in addition to changes in gene expression, the transition to totipotency in the plant zygote is accompanied by resetting of the epigenetic reprogramming that occurred during gamete formation.
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Bhandare, Vishwambhar, and Amutha Ramaswamy. "Structural Dynamics of Human Argonaute2 and Its Interaction with siRNAs Designed to Target Mutant tdp43." Advances in Bioinformatics 2016 (March 6, 2016): 1–13. http://dx.doi.org/10.1155/2016/8792814.
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The human Argonaute2 protein (Ago2) is a key player in RNA interference pathway and small RNA recognition by Ago2 is the crucial step in siRNA mediated gene silencing mechanism. The present study highlights the structural and functional dynamics of human Ago2 and the interaction mechanism of Ago2 with a set of seven siRNAs for the first time. The human Ago2 protein adopts two conformations such as “open” and “close” during the simulation of 25 ns. One of the domains named as PAZ, which is responsible for anchoring the 3′-end of siRNA guide strand, is observed as a highly flexible region. The interaction between Ago2 and siRNA, analyzed using a set of siRNAs (targeting at positions 128, 251, 341, 383, 537, 1113, and 1115 of mRNA) designed to target tdp43 mutants causing Amyotrophic Lateral Sclerosis (ALS) disease, revealed the stable and strong recognition of siRNA by the Ago2 protein during dynamics. Among the studied siRNAs, the siRNA341 is identified as a potent siRNA to recognize Ago2 and hence could be used further as a possible siRNA candidate to target the mutant tdp43 protein for the treatment of ALS patients.
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Mainini, Francesco, and Michael R. Eccles. "Lipid and Polymer-Based Nanoparticle siRNA Delivery Systems for Cancer Therapy." Molecules 25, no. 11 (June 10, 2020): 2692. http://dx.doi.org/10.3390/molecules25112692.
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RNA interference (RNAi) uses small interfering RNAs (siRNAs) to mediate gene-silencing in cells and represents an emerging strategy for cancer therapy. Successful RNAi-mediated gene silencing requires overcoming multiple physiological barriers to achieve efficient delivery of siRNAs into cells in vivo, including into tumor and/or host cells in the tumor micro-environment (TME). Consequently, lipid and polymer-based nanoparticle siRNA delivery systems have been developed to surmount these physiological barriers. In this article, we review the strategies that have been developed to facilitate siRNA survival in the circulatory system, siRNA movement from the blood into tissues and the TME, targeted siRNA delivery to the tumor or specific cell types, cellular uptake, and escape from endosomal degradation. We also discuss the use of various types of lipid and polymer-based carriers for cancer therapy, including a section on anti-tumor nanovaccines enhanced by siRNAs. Finally, we review current and recent clinical trials using NPs loaded with siRNAs for cancer therapy. The siRNA cancer therapeutics field is rapidly evolving, and it is conceivable that precision cancer therapy could, in the relatively near future, benefit from the combined use of cancer therapies, for example immune checkpoint blockade together with gene-targeting siRNAs, personalized for enhancing and fine-tuning a patient’s therapeutic response.
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Zhang, Xueli, Amu Gubu, Jianfei Xu, Ning Yan, Wenbo Su, Di Feng, Qian Wang, and Xinjing Tang. "Tetrazine-Induced Bioorthogonal Activation of Vitamin E-Modified siRNA for Gene Silencing." Molecules 27, no. 14 (July 8, 2022): 4377. http://dx.doi.org/10.3390/molecules27144377.
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The temporal activation of siRNA provides a valuable strategy for the regulation of siRNA activity and conditional gene silencing. The bioorthogonal bond-cleavage reaction of benzonorbonadiene and tetrazine is a promising trigger in siRNA temporal activation. Here, we developed a new method for the bio-orthogonal chemical activation of siRNA based on the tetrazine-induced bond-cleavage reaction. Small-molecule activatable caged siRNAs were developed with the 5′-vitamin E-benzonobonadiene-modified antisense strand targeting the green fluorescent protein (GFP) gene and the mitotic kinesin-5 (Eg5) gene. The addition of tetrazine triggered the reaction with benzonobonadiene linker and induced the linker cleavage to release the active siRNA. Additionally, the conditional gene silencing of both exogenous GFP and endogenous Eg5 genes was successfully achieved with 5′-vitamin E-benzonobonadiene-caged siRNAs, which provides a new uncaging strategy with small molecules.
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Jagannath, Aarti, and Matthew J. A. Wood. "Localization of Double-stranded Small Interfering RNA to Cytoplasmic Processing Bodies Is Ago2 Dependent and Results in Up-Regulation of GW182 and Argonaute-2." Molecular Biology of the Cell 20, no. 1 (January 2009): 521–29. http://dx.doi.org/10.1091/mbc.e08-08-0796.
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Processing bodies (P-bodies) are cytoplasmic foci implicated in the regulation of mRNA translation, storage, and degradation. Key effectors of microRNA (miRNA)-mediated RNA interference (RNAi), such as Argonaute-2 (Ago2), miRNAs, and their cognate mRNAs, are localized to these structures; however, the precise role that P-bodies and their component proteins play in small interfering RNA (siRNA)-mediated RNAi remains unclear. Here, we investigate the relationship between siRNA-mediated RNAi, RNAi machinery proteins, and P-bodies. We show that upon transfection into cells, siRNAs rapidly localize to P-bodies in their native double-stranded conformation, as indicated by fluorescence resonance energy transfer imaging and that Ago2 is at least in part responsible for this siRNA localization pattern, indicating RISC involvement. Furthermore, siRNA transfection induces up-regulated expression of both GW182, a key P-body component, and Ago2, indicating that P-body localization and interaction with GW182 and Ago2 are important in siRNA-mediated RNAi. By virtue of being centers where these proteins and siRNAs aggregate, we propose that the P-body microenvironment, whether as microscopically visible foci or submicroscopic protein complexes, facilitates siRNA processing and siRNA-mediated silencing through the action of its component proteins.
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Gao, Yong, Michael A. Lobritz, Justin Roth, Measho Abreha, Kenneth N. Nelson, Immaculate Nankya, Dawn M. Moore-Dudley, Awet Abraha, Stanton L. Gerson, and Eric J. Arts. "Targets of Small Interfering RNA Restriction during Human Immunodeficiency Virus Type 1 Replication." Journal of Virology 82, no. 6 (January 16, 2008): 2938–51. http://dx.doi.org/10.1128/jvi.02126-07.
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ABSTRACT Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5α did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5′ of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3′-to-5′ siRNA amplification and spreading. In contrast, HIV-1 RNA 3′ of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively “naked” cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5α, or other host restriction factors.
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Grover, Jeffrey W., Diane Burgess, Timmy Kendall, Abdul Baten, Suresh Pokhrel, Graham J. King, Blake C. Meyers, Michael Freeling, and Rebecca A. Mosher. "Abundant expression of maternal siRNAs is a conserved feature of seed development." Proceedings of the National Academy of Sciences 117, no. 26 (June 15, 2020): 15305–15. http://dx.doi.org/10.1073/pnas.2001332117.
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Small RNAs are abundant in plant reproductive tissues, especially 24-nucleotide (nt) small interfering RNAs (siRNAs). Most 24-nt siRNAs are dependent on RNA Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). InBrassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here, we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression duringB. rapaseed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together, these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.
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Chen, Changmai, Nannan Jing, Zhongyu Wang, Yu Zhang, Wei Chen, and Xinjing Tang. "Multimerized self-assembled caged two-in-one siRNA nanoparticles for photomodulation of RNAi-induced gene silencing." Chemical Science 11, no. 45 (2020): 12289–97. http://dx.doi.org/10.1039/d0sc03562a.
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Paul, Amber M., Yongliang Shi, Dhiraj Acharya, Jessica R. Douglas, Amanda Cooley, John F. Anderson, Faqing Huang, and Fengwei Bai. "Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro." Journal of General Virology 95, no. 8 (August 1, 2014): 1712–22. http://dx.doi.org/10.1099/vir.0.066084-0.
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Dengue virus (DENV) infection in humans can cause flu-like illness, life-threatening haemorrhagic fever or even death. There is no specific anti-DENV therapeutic or approved vaccine currently available, partially due to the possibility of antibody-dependent enhancement reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are vulnerable to degradation by serum nucleases and rapid renal excretion due to their small size and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that cationic AuNP–siRNA complexes could enter Vero cells and significantly reduce DENV serotype 2 (DENV-2) replication and infectious virion release under both pre- and post-infection conditions. In addition, RNase-treated AuNP–siRNA complexes could still inhibit DENV-2 replication, suggesting that AuNPs maintained siRNA stability. Collectively, these results demonstrated that AuNPs were able to efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy.
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Zheng, Bo-jian, Yi Guan, Qingquan Tang, Du Cheng, Frank Y. Xie, Ming-Liang He, Kwok-Wah Chan, et al. "Prophylactic and Therapeutic Effects of Small Interfering Rna Targeting Sars-Coronavirus." Antiviral Therapy 9, no. 3 (April 1, 2003): 365–74. http://dx.doi.org/10.1177/135965350400900310.
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Objectives To identify and characterize the siRNA duplexes that are effective for inhibition of SARS-CoV infection and replication in the non-human primate cells. This in vitro study will serve as the foundation for development of novel anti-SARS therapeutics. Methods 48 siRNA sequences were designed for targeting regions throughout entire SARS-CoV genome RNA including open-reading frames for several key proteins. Chemically synthesized siRNA duplexes were transfected into foetal rhesus kidney (FRhK-4) cells prior to or after SARS-CoV infection. The inhibitory effects of the siRNAs were evaluated for reductions of intracellular viral genome copy number and viral titres in the cell culture medium measured by Q-RT-PCR and CPE-based titration, respectively. Four siRNA duplexes were found to achieve potent inhibition of SARS-CoV infection and replication. A prolonged prophylactic effect of siRNA duplexes with up to 90% inhibition that lasted for at least 72 h was observed. Combination of active siRNA duplexes targeting different regions of the viral genome resulted in therapeutic activity of up to 80% inhibition. Conclusion Chemically synthesized siRNA duplexes targeting SARS-CoV genomic RNA are potent agents for inhibition of the viral infection and replication. The location effects of siRNAs were revealed at both genome sequence and open-reading frame levels. The rapid development of siRNA-based SARS-CoV inhibitors marked a novel approach for combating newly emergent infectious diseases.
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Wang, Xuan, and Fa Zhang. "Prediction of siRNA Efficacy Using BP Neural Network." Applied Mechanics and Materials 644-650 (September 2014): 5341–45. http://dx.doi.org/10.4028/www.scientific.net/amm.644-650.5341.
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In the last decade, RNA interference (RNAi) by small interfering RNAs (siRNAs) has become a hot topic in both molecular biology and bioinformatics. The success of RNAi gene silencing depends on the specificity of siRNAs for particular mRNA sequences. As a targeted gene could have thousands of potential siRNAs, finding the most efficient siRNAs among them constitutes a huge challenge. Previous studies such as rules scoring or machine learning aim to optimize the selection of target siRNAs. However, these methods have low accuracy or poor generalization ability, when they used new datasets to test. In this study, a siRNA efficacy prediction method using BP neural network (BP-GA) was proposed. For more efficient siRNA candidate prediction, twenty rational design rules our defined were used to filter siRNA candidate and they were used in the neural network model as input parameters. Furthermore, the performance optimization of network model has been done by using genetic algorithm and setting optimal training parameters. The BP-GA was trained on 2431 siRNA records and tested using a new public dataset. Compared with existing rules scoring and BP methods, BP-GA has higher prediction accuracy and better generalization ability.
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Brown, Christopher R., Swati Gupta, June Qin, Timothy Racie, Guo He, Scott Lentini, Ryan Malone, et al. "Investigating the pharmacodynamic durability of GalNAc–siRNA conjugates." Nucleic Acids Research 48, no. 21 (August 18, 2020): 11827–44. http://dx.doi.org/10.1093/nar/gkaa670.
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Abstract One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc–siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc–siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.
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Peng, Jie, Jiahui Ma, Xue Yang, Huan He, Haopeng Wu, Tongtong Ma, and Jianhua Lu. "Water-Soluble Polymer Assists N-Methyl-D-Aspartic Acid Receptor 2B siRNA Delivery to Relieve Chronic Inflammatory Pain In Vitro and In Vivo." Pain Research and Management 2018 (2018): 1–9. http://dx.doi.org/10.1155/2018/7436060.
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We constructed a water-soluble lipopolymer (WSLP) as a nonviral gene carrier to deliver siRNA targeting NR2B. The cytotoxicity and serum stability of WSLP loaded with siRNA were evaluated, and the knockdown efficiency of WSLP/NR2B-siRNA in PC12 cells was examined. The results showed that WSLP could protect the loading siRNAs from enzymatic degradation in serum and exhibit low cytotoxicity to cells. After transfection, WSLP/NR2B-siRNA complexes reduced the NR2B transcriptional level by 50% and protein level by 55% compared to control siRNA. Moreover, 3 days after intrathecal injection of WSLP/NR2B-siRNA complexes into rats, the NR2B protein expression decreased significantly to 58%, compared to control treatment (p<0.01). Injection of WSLP with scrambled siRNA or of polyethylenimine (PEI) with NR2B-siRNA did not show this inhibitory effect. Additionally, injection of WSLP/NR2B-siRNA complexes significantly relieved inflammatory pain in rats at 3, 4, and 5 days with reduced MWT and decreased TWL scores, while injection of WSLP with scrambled siRNA or of PEI with NR2B-siRNA did not. These results demonstrated that WSLP can efficiently deliver siRNA targeting NR2B to PC12 cells and relieve pain in rats with chronic inflammatory pain.
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Sattarivand, Masoumeh, and Elahe Aliheydari. "siRNA and apoptosis in breast cancer cells: A minireview." Journal of Biological Studies 6, no. 1 (May 10, 2023): 123–36. http://dx.doi.org/10.62400/jbs.v6i1.7769.
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Small interfering RNA (siRNA), is a class of double-stranded RNA at first non-coding RNA molecules, operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. siRNAs have been widely used to study gene function and extensively exploited for their potential therapeutic applications. siRNAs have gained attention as a potential therapeutic reagent due to their ability to inhibit specific genes in cancer cells. Increased resistance to apoptosis is a challenging issue for treatment of many cancers, including breast cancer. It has been recently reported that siRNAs and RNAi technology can be used to increase the apoptotic susceptibility of cancer cells. It has been shown that apoptosis is induced in cancer cells by siRNA-mediated silencing of the livin/ML-IAP/KIAP gene. Association of siRNA with apoptosis via mitochondrial depolarization and caspase-3 activation has been highlighted in cancer cells. Although many aspects of siRNA actions in cancer cells have been revealed through in vivo and in vitro studies, the biological mechanisms underlying siRNA mediated knockdown of gene expression and apoptosis induction in cancer cells are not yet fully understood. The main aim of this review is to investigate the effects of siRNAs on apoptosis induction in breast cancer cells.
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Vigh, Maria L., Simon Bressendorff, Axel Thieffry, Laura Arribas-Hernández, and Peter Brodersen. "Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis." Nucleic Acids Research 50, no. 3 (January 17, 2022): 1396–415. http://dx.doi.org/10.1093/nar/gkab1289.
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Abstract Amplification of short interfering RNA (siRNAs) via RNA-dependent RNA polymerases (RdRPs) is of fundamental importance in RNA silencing. Plant microRNA (miRNA) action generally does not involve engagement of RdRPs, in part thanks to a poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that inactivation of the exosome subunit RRP45B and SKI2 results in similar patterns of miRNA-induced siRNA production. Furthermore, loss of the nuclear exosome adaptor HEN2 leads to secondary siRNA production from miRNA targets largely distinct from those producing siRNAs in ski2. Importantly, mutation of the Release Factor paralogue PELOTA1 required for subunit dissociation of stalled ribosomes causes siRNA production from miRNA targets overlapping with, but distinct from, those affected in ski2 and rrp45b mutants. We also show that in exosome mutants, miRNA targets can be sorted into producers and non-producers of illicit secondary siRNAs based on trigger miRNA levels and miRNA:target affinity rather than on presence of 5′-cleavage fragments. We propose that stalled RNA-Induced Silencing Complex (RISC) and ribosomes, but not mRNA cleavage fragments released from RISC, trigger siRNA production, and that the exosome limits siRNA amplification by reducing RISC dwell time on miRNA target mRNAs while PELOTA1 does so by reducing ribosome stalling.
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Marine, Shane, Amit Bahl, Marc Ferrer, and Eugen Buehler. "Common Seed Analysis to Identify Off-Target Effects in siRNA Screens." Journal of Biomolecular Screening 17, no. 3 (November 15, 2011): 370–78. http://dx.doi.org/10.1177/1087057111427348.
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Genome-scale small interfering RNA (siRNA) screens have become an increasingly popular approach to new target identification and pathway elucidation. However, the large data sets generated from siRNA screens have demonstrated high false-positive rates and the requirement for extensive experimental triage to distinguish true hits. A number of groups have independently reported the presence of siRNAs with identical seed sequences among their top screening hits. Based on these observations, we have developed a comprehensive technique for detecting and visualizing seed-based off-target effects in siRNA screening data. This is accomplished by analyzing the behavior of siRNAs that share identical seed sequences, which we refer to as common seed analysis (CSA). By applying these techniques to primary screening data of the Wnt pathway, we identify 158 distinct seed sequences that have a statistically significant effect on the assay. The promiscuous seed sequences identified in this manner can then be discounted in the analysis of follow-up experiments using single siRNAs. The ability to detect off-target effects when sufficient numbers of siRNAs share a common seed has significant implications for the design of siRNA screening experiments, data analysis, hit selection, and library design.
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Zhang, Zhanhui, Sachin Teotia, Jihua Tang, and Guiliang Tang. "Perspectives on microRNAs and Phased Small Interfering RNAs in Maize (Zea mays L.): Functions and Big Impact on Agronomic Traits Enhancement." Plants 8, no. 6 (June 12, 2019): 170. http://dx.doi.org/10.3390/plants8060170.
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Small RNA (sRNA) population in plants comprises of primarily micro RNAs (miRNAs) and small interfering RNAs (siRNAs). MiRNAs play important roles in plant growth and development. The miRNA-derived secondary siRNAs are usually known as phased siRNAs, including phasiRNAs and tasiRNAs. The miRNA and phased siRNA biogenesis mechanisms are highly conserved in plants. However, their functional conservation and diversification may differ in maize. In the past two decades, lots of miRNAs and phased siRNAs have been functionally identified for curbing important maize agronomic traits, such as those related to developmental timing, plant architecture, sex determination, reproductive development, leaf morphogenesis, root development and nutrition, kernel development and tolerance to abiotic stresses. In contrast to Arabidopsis and rice, studies on maize miRNA and phased siRNA biogenesis and functions are limited, which restricts the small RNA-based fundamental and applied studies in maize. This review updates the current status of maize miRNA and phased siRNA mechanisms and provides a survey of our knowledge on miRNA and phased siRNA functions in controlling agronomic traits. Furthermore, improvement of those traits through manipulating the expression of sRNAs or their targets is discussed.
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Karunanithi, Sivarajan, Vidya Oruganti, Simone Marker, Angela M. Rodriguez-Viana, Franziska Drews, Marcello Pirritano, Karl Nordström, Martin Simon, and Marcel H. Schulz. "Exogenous RNAi mechanisms contribute to transcriptome adaptation by phased siRNA clusters in Paramecium." Nucleic Acids Research 47, no. 15 (June 28, 2019): 8036–49. http://dx.doi.org/10.1093/nar/gkz553.
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Abstract Extensive research has characterized distinct exogenous RNAi pathways interfering in gene expression during vegetative growth of the unicellular model ciliate Paramecium. However, role of RNAi in endogenous transcriptome regulation, and environmental adaptation is unknown. Here, we describe the first genome-wide profiling of endogenous sRNAs in context of different transcriptomic states (serotypes). We developed a pipeline to identify, and characterize 2602 siRNA producing clusters (SRCs). Our data show no evidence that SRCs produce miRNAs, and in contrast to other species, no preference for strand specificity of siRNAs. Interestingly, most SRCs overlap coding genes and a separate group show siRNA phasing along the entire open reading frame, suggesting that the mRNA transcript serves as a source for siRNAs. Integrative analysis of siRNA abundance and gene expression levels revealed surprisingly that mRNA and siRNA show negative as well as positive associations. Two RNA-dependent RNA Polymerase mutants, RDR1 and RDR2, show a drastic loss of siRNAs especially in phased SRCs accompanied with increased mRNA levels. Importantly, most SRCs depend on both RDRs, reminiscent to primary siRNAs in the RNAi against exogenous RNA, indicating mechanistic overlaps between exogenous and endogenous RNAi contributing to flexible transcriptome adaptation.
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Wilson, Joyce A., and Christopher D. Richardson. "Hepatitis C Virus Replicons Escape RNA Interference Induced by a Short Interfering RNA Directed against the NS5b Coding Region." Journal of Virology 79, no. 11 (June 1, 2005): 7050–58. http://dx.doi.org/10.1128/jvi.79.11.7050-7058.2005.
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ABSTRACT RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). We have shown previously that double-stranded siRNA molecules designed to target the HCV genome block gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. However, we now show that this block is not complete. After several treatments with a highly effective siRNA, we have shown growth of replicon RNAs that are resistant to subsequent treatment with the same siRNA. However, these replicon RNAs were not resistant to siRNA targeting another part of the genome. Sequence analysis of the siRNA-resistant replicons showed the generation of point mutations within the siRNA target sequence. In addition, the use of a combination of two siRNAs together severely limited escape mutant evolution. This suggests that RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.
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Sohrab, Sayed Sartaj, Sherif Aly El-Kafrawy, Zeenat Mirza, Mohammad Amjad Kamal, and Esam Ibraheem Azhar. "Design and Delivery of Therapeutic siRNAs: Application to MERS-Coronavirus." Current Pharmaceutical Design 24, no. 1 (March 22, 2018): 62–77. http://dx.doi.org/10.2174/1381612823666171109112307.
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Background: The MERS-CoV is a novel human coronavirus causing respiratory syndrome since April 2012. The replication of MERS-CoV is mediated by ORF 1ab and viral gene activity can be modulated by RNAi approach. The inhibition of virus replication has been documented in cell culture against multiple viruses by RNAi approach. Currently, very few siRNA against MERS-CoV have been computationally designed and published. Methods: In this review, we have discussed the computational designing and delivery of potential siRNAs. Potential siRNA can be designed to silence a desired gene by considering many factors like target site, specificity, length and nucleotide content of siRNA, removal of potential off-target sites, toxicity and immunogenic responses. The efficient delivery of siRNAs into targeted cells faces many challenges like enzymatic degradation and quick clearance through renal system. The siRNA can be delivered using transfection, electroporation and viral gene transfer. Currently, siRNAs delivery has been improved by using advanced nanotechnology like lipid nanoparticles, inorganic nanoparticles and polymeric nanoparticles. Conclusion: The efficacy of siRNA-based therapeutics has been used not only against many viral diseases but also against non-viral diseases, cancer, dominant genetic disorders, and autoimmune disease. This innovative technology has attracted researchers, academia and pharmaceuticals industries towards designing and development of highly effective and targeted disease therapy. By using this technology, effective and potential siRNAs can be designed, delivered and their efficacy with toxic effects and immunogenic responses can be tested against MERS-CoV.
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Neugebauer, Maximilian, Clara E. Grundmann, Michael Lehnert, Felix von Stetten, Susanna M. Früh, and Regine Süss. "Analyzing siRNA Concentration, Complexation and Stability in Cationic Dendriplexes by Stem-Loop Reverse Transcription-qPCR." Pharmaceutics 14, no. 7 (June 25, 2022): 1348. http://dx.doi.org/10.3390/pharmaceutics14071348.
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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.
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Cejka, Daniel, Doris Losert, and Volker Wacheck. "Short interfering RNA (siRNA): tool or therapeutic?" Clinical Science 110, no. 1 (December 12, 2005): 47–58. http://dx.doi.org/10.1042/cs20050162.
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Gene silencing by siRNA (short interfering RNA) is a still developing field in biology and has evolved as a novel post-transcriptional gene silencing strategy with therapeutic potential. With siRNAs, virtually every gene in the human genome contributing to a disease becomes amenable to regulation, thus opening unprecedented opportunities for drug discovery. Besides the well-established role for siRNA as a tool for target screening and validation in vitro, recent progress of siRNA delivery in vivo raised expectations for siRNA drugs as the up-and-coming ‘magic bullet’. Whether siRNA compounds will make it as novel chemical entities from ‘bench to bedside’ will probably depend largely on improving their pharmacokinetics in terms of plasma stability and cellular uptake. Whereas locally administered siRNAs have already entered the first clinical trials, strategies for successful systemic delivery of siRNA are still in a preclinical stage of development. Irrespective of its therapeutic potential, RNAi (RNA interference) has unambiguously become a valuable tool for basic research in biology and thereby it will continue to have a major impact on medical science. In this review, we will give a brief overview about the history and current understanding of RNAi and focus on potential applications, especially as a therapeutic option to treat human disease.
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Jing, Jiaona, Ping Li, Tiejun Li, Yuncheng Sun, and Huaijin Guan. "RNA Interference Targeting Connective Tissue Growth Factor Inhibits the Transforming Growth Factor-β2Induced Proliferation in Human Tenon Capsule Fibroblasts." Journal of Ophthalmology 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/354798.
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Purpose. This study was to determine the effect of CTGF-small interfering RNA (siRNA) on TGF-β2-induced proliferation in human Tenon capsule fibroblasts (HTFs).Methods. HTFs were transfected with four of CTGF-siRNAs separately for screening of gene silencing efficacy that was determined by transcript level measured by quantitative real-time PCR (qRT-PCR). Recombinant TGF-β2was added into the culture to stimulate the proliferation of HTFs. The gene silencing efficacy of the siRNAs was evaluated by qRT-PCR and immunofluorescence of CTGF transcript and protein levels. The viability of HTFs was determined by cell counting kit-8 (CCK-8). FCM was used to assess cell cycle after CTGF-siRNA transfection.Results. The expression of CTGF and proliferation of HTFs were increased significantly by TGF-β2stimulation. The transfection of CTGF-siRNA abolished the upregulation of CTGF and cell proliferation induced by TGF-β2. The analysis of cell cycle indicated that CTGF-siRNA treatment stimulated cells from S phase to G0/G1 phase in comparison with the inverse physiologic function of TGF-β2.Conclusion. CTGF targeting siRNA could effectively suppress the expression of CTGF and attenuate the proliferation of HTFs. The siRNA approach may provide a therapeutic option for eliminating filtration bleb scarring after glaucoma filtration surgery (GFS).
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Ramachandiran, Sampath, Arsene Adone, Xiangxue Guo, Albert Liao, Uston Robert Sunay, Lee Ratner, Haian Fu, Izidore S. Lossos, Harold Saavedra, and Leon Bernal-Mizrachi. "Genomic Stability: A Novel Function of NF-Kb in Lymphomas." Blood 114, no. 22 (November 20, 2009): 3240. http://dx.doi.org/10.1182/blood.v114.22.3240.3240.
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Abstract Abstract 3240 Poster Board III-177 Lymphomas are heterogeneous diseases comprising multiple clinical and biological subgroups. Several studies have shown that genomic instability and constitutive activation of the NF-kB pathway are key features for lymphoma development (Shen M,Hematologica 2007). However, it remains unclear whether changes in activation of the canonical and non-canonical NF-kB pathways (rel-A/p50 and rel-B/p52, respectively) reflect a response to genomic instability and therefore promotes lymphomagenesis. To answer this question, we first demonstrated in two lymphoma cell lines (Daudi and OCI-Ly3) that DNA damage induced by Doxorubicin (2 mcg/mL) resulted in nuclear localization of rel-A and Rel-B. Then, to determine the role of each NF-kB pathway in DNA repair and centrosome duplication we compared the number of cells positive for phospo-H2aX (pH2aX) and centrosomes numbers (measured by gamma-tubulin) in p105-siRNA (canonical) and P100-siRNA (non-canonical) with luciferase-siRNA (control) expressing cells. Our results showed that the expression of p105 and p100 siRNAs increase the number of pH2aX (+) cells compared to control. Subsequently, a time course measuring pH2aX (+) cells was performed after treating p105, P100 and luciferase siRNA OCI-ly3 expressing cell lines with Doxorubicin (2 mcg/mL). In cells expressing luciferase siRNA, pH2aX (+) cells peak (60%) at 60 minutes (min) and return to normal at 120 min, in p105 siRNA –cells pH2aX peak at 90 min (90% + cells) and then decrease similarly to luciferase siRNA cells. P100 siRNA cells demonstrate a continuous increased in pH2aX (+) cells up to 80%. In addition, p100 siRNA expression was associated with centrosome amplification (>2 centrosomes in 20-30% of the cells vs. < 8% in p105 or Luciferase siRNA expressing cells). Also, the expression of NF-kB siRNAs delayed doxorubicin-induced phosphorylation of p53 (serine 15 – target of ATM) and CHK2. To evaluate whether the genomic instability caused by both NF-kB siRNAs affects tumor development, we performed xenograft experiments. Our results demonstrated that NF-kB siRNAs not only slow down tumor initiation but prevented tumor development (p105 siRNA= 8 days delayed and 22% were tumor free and p100 siRNA= 13.5 days delayed and 55% were tumor free compared to luciferase siRNA). To investigate these findings in primary tissues we measured the number of phospo-H2aX (+) cells and the levels of rel-A and rel-B nuclear localization in 40 primary lymphoma tumor samples. Our results demonstrated that phospo-H2aX levels inversely correlated with rel-B nuclear localization (r=-0.58, p<0.0001). To identify possible explanations for these results, gene expression analysis was performed in cells expressing NF-kB siRNAs. Our results demonstrated that p105 siRNA regulated genes involved in DNA repair (PPP2R5C, ING5, SYF2, SYF2, XRCC6, etc) and p100 siRNA regulated genes involved in both DNA repair and centrosome duplication (GADD45 alpha, cyclin G, REDD1, PCBP4, etc) consistent with our results above. Quantitative PCR for some these genes during a doxorubicin-time course confirmed GADD45 alpha, cyclin G, PCBP4 and SFRS6 to be induced. We explored further the role of GADD45 alpha in lymphomas and found that knock down of this protein increase doxorubicin sensitivity by 50-fold. Overall this study demonstrated that activation of each NF-kB pathway is essential for maintaining genomic stability and therefore promoting tumor resistance to chemotherapy in lymphomas. In addition, we identified that GADD45 alpha is important target of the non-canonical NF-kB pathway for mediating genomic stability. These findings provide the rationale for designing novel agents aiming at targeting key genes involved in genomic stability. Disclosures No relevant conflicts of interest to declare.
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Zhao, Zhen, Yuanke Li, Hao Liu, Akshay Jain, Pratikkumar Vinodchandra Patel, and Kun Cheng. "Co-delivery of IKBKE siRNA and cabazitaxel by hybrid nanocomplex inhibits invasiveness and growth of triple-negative breast cancer." Science Advances 6, no. 29 (July 2020): eabb0616. http://dx.doi.org/10.1126/sciadv.abb0616.
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IKBKE is an oncogene in triple-negative breast cancer (TNBC), and we demonstrate that IKBKE small interfering RNA (siRNA) inhibits the proliferation, migration, and invasion of TNBC cells. Despite the recent success of siRNA therapeutics targeting to the liver, there still remains a great challenge to deliver siRNAs to solid tumors. Here, we report a hybrid nanocomplex to co-deliver the IKBKE siRNA and cabazitaxel to TNBC to achieve an optimal antitumor effect. The nanocomplex is modified with hyaluronic acid to target CD44 on TNBC cells. The nanocomplex shows higher cellular uptake and better tumor penetration of the encapsulated cargos. The nanocomplex also exhibits high tumor accumulation and antitumor activity in an orthotopic TNBC mouse model. Encapsulation of cabazitaxel in the nanocomplex enhances the activity of the IKBKE siRNA. The hybrid nanocomplex provides a novel and versatile platform for combination therapies using siRNAs and chemotherapy.
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Yang, Peizhen, Ericka Havecker, Matthew Bauer, Carl Diehl, Bill Hendrix, Paul Hoffer, Timothy Boyle, John Bradley, Amy Caruano-Yzermans, and Jill Deikman. "Beyond identity: Understanding the contribution of the 5’ nucleotide of the antisense strand to RNAi activity." PLOS ONE 16, no. 9 (September 7, 2021): e0256863. http://dx.doi.org/10.1371/journal.pone.0256863.
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In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene’s mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5’ nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5’ nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5’ nt identities and different pairing states between the 5’ antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5’ nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5’ thermodynamic rules governing topical siRNA efficacy across plants and animals.
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Fookolaee, Sakineh Poorhosein, Samad Karkhah, Mahdiye Saadi, Subho Majumdar, and Ahmad Karkhah. "Novel Computational Approaches to Developing Potential STAT4 Silencing siRNAs for Immunomodulation of Atherosclerosis." Current Computer-Aided Drug Design 16, no. 5 (November 9, 2020): 599–604. http://dx.doi.org/10.2174/1573409915666191018125653.
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Background: Small interfering RNAs (siRNAs) are known as commonly used targeting mRNAs tools for suppressing gene expression. Since Signal Transducer and Activator of Transcription 4 (STAT4) is considered as a significant transcription factor for generation and differentiation of Th1 cells during vascular dysfunction and atherosclerosis, suppressing STAT4 could represent novel immunomodulatory therapies against atherosclerosis. Objective: Therefore, the current study was conducted to design efficient siRNAs specific for STAT4 and to evaluate different criteria affecting their functionality. Methods: In the present study, all related sequences of STAT4 gene were retrieved from Gen Bank database. Multiple sequence alignment was carried out to recognize Open Reading Frame (ORF) and conserved region. Then, siDirect 2.0 server was applied for the development of candidate siRNA molecules and confirmation of predicted molecules was performed using Dharma siRNA technology and GeneScript siRNA targetfinder. In addition, BLAST tool was used against whole Genebank databases to identify potential off-target genes. DNA/RNA GC content calculator and mfold server were used to calculate GC content and secondary structure prediction of designed siRNA, respectively. Finally, IntaRNA program was used to study the thermodynamics of interaction between predicted siRNA and target gene. Results: Based on the obtained results, three efficient siRNA molecules were designed and validated for STAT4 gene silencing using computational methods, which may result in suppressing STAT4 gene expression. Conclusion: According to our results, this study shows that siRNA targeting STAT4 can be considered as a therapeutic agent in many Th1-mediated pathologic conditions specially atherosclerosis.
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Navalikhina, A. G., M. Z. Antonyuk, and T. K. Ternovska. "The role of siRNAs in genome stability maintaining in the bread wheat introgression lines." Faktori eksperimental'noi evolucii organizmiv 26 (September 1, 2020): 108–13. http://dx.doi.org/10.7124/feeo.v26.1251.
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Aim. Deviations of the siRNAs levels from the parental ones in the plants with hybrid genomes are associated with the activation of transposable elements (TE). This, in turn, lead to the further genome rearrangements. Introgression lines Triticum aestivum / Amblyopyrum muticum are cytologically stable, however, there are visible signs of genetic and (or) epigenetic restructuring that are still going on. Molecular mechanisms of these processes are the subject of our study. Methods. The levels of siRNAs in the plant lemmas were determined by small RNA-seq. Reads of the small RNA libraries were aligned to the repeats to find siRNA sequences. Results. Introgression lines (ILs) and parental amphidiploid have variable levels of siRNAs regulating MITE and CACTA transposable elements, compared with the parental bread wheat variety. For twelve TE sequences, majority of which are CACTA elements, decrease in the levels of siRNAs in ILs and amphidiploid, compared to the wheat, is statistically significant. Decreased siRNAs levels could lead to the activation of corresponding TE classes. Conclusions. Variation of siRNA levels in ILs and amphidiploid can be the key factor that cause rearrangements in their genomes. These include activation of TEs, changes in DNA methylation patterns, and gene expression variation. Therefore, detected changes in siRNA levels can be the molecular mechanisms of the processes that occur in studied hybrid genomes.
Keywords: siRNA, transposable elements, amphidiploid, introgression lines.
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Kirkbride, Ryan C., Jie Lu, Changqing Zhang, Rebecca A. Mosher, David C. Baulcombe, and Z. Jeffrey Chen. "Maternal small RNAs mediate spatial-temporal regulation of gene expression, imprinting, and seed development in Arabidopsis." Proceedings of the National Academy of Sciences 116, no. 7 (January 28, 2019): 2761–66. http://dx.doi.org/10.1073/pnas.1807621116.
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Arabidopsis seed development involves maternal small interfering RNAs (siRNAs) that induce RNA-directed DNA methylation (RdDM) through the NRPD1-mediated pathway. To investigate their biological functions, we characterized siRNAs in the endosperm and seed coat that were separated by laser-capture microdissection (LCM) in reciprocal genetic crosses with an nrpd1 mutant. We also monitored the spatial-temporal activity of the NRPD1-mediated pathway on seed development using the AGO4:GFP::AGO4 (promoter:GFP::protein) reporter and promoter:GUS sensors of siRNA-mediated silencing. From these approaches, we identified four distinct groups of siRNA loci dependent on or independent of the maternal NRPD1 allele in the endosperm or seed coat. A group of maternally expressed NRPD1-siRNA loci targets endosperm-preferred genes, including those encoding AGAMOUS-LIKE (AGL) transcription factors. Using translational promoter:AGL::GUS constructs as sensors, we demonstrate that spatial and temporal expression patterns of these genes in the endosperm are regulated by the NRPD1-mediated pathway irrespective of complete silencing (AGL91) or incomplete silencing (AGL40) of these target genes. Moreover, altered expression of these siRNA-targeted genes affects seed size. We propose that the corresponding maternal siRNAs could account for parent-of-origin effects on the endosperm in interploidy and hybrid crosses. These analyses reconcile previous studies on siRNAs and imprinted gene expression during seed development.
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Sajid, Muhammad Imran, Muhammad Moazzam, Shun Kato, Kayley Yeseom Cho, and Rakesh Kumar Tiwari. "Overcoming Barriers for siRNA Therapeutics: From Bench to Bedside." Pharmaceuticals 13, no. 10 (October 7, 2020): 294. http://dx.doi.org/10.3390/ph13100294.
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The RNA interference (RNAi) pathway possesses immense potential in silencing any gene in human cells. Small interfering RNA (siRNA) can efficiently trigger RNAi silencing of specific genes. FDA Approval of siRNA therapeutics in recent years garnered a new hope in siRNA therapeutics. However, their therapeutic use is limited by several challenges. siRNAs, being negatively charged, are membrane-impermeable and highly unstable in the systemic circulation. In this review, we have comprehensively discussed the extracellular barriers, including enzymatic degradation of siRNAs by serum endonucleases and RNAases, rapid renal clearance, membrane impermeability, and activation of the immune system. Besides, we have thoroughly described the intracellular barriers such as endosomal trap and off-target effects of siRNAs. Moreover, we have reported most of the strategies and techniques in overcoming these barriers, followed by critical comments in translating these molecules from bench to bedside.
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Ghaziani, Tahereh, Ying Shan, Richard W. Lambrecht, and Herbert L. Bonkovsky. "Synergistic Up-Regulation of Heme Oxygenase-1 in Human Liver by Heme and siRNA to Bach1:Possible Therapeutic Implications." Blood 104, no. 11 (November 16, 2004): 1633. http://dx.doi.org/10.1182/blood.v104.11.1633.1633.
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Abstract Background: Heme oxygenase-1 (HO-1) is an antioxidant defense enzyme that converts toxic heme into antioxidants. HO-1 is strongly up-regulated by its physiologic substrate, heme, which is currently the treatment of choice for acute attacks of porphyria and which may have other therapeutic uses, as well (e.g., for cytoprotection or amelioration of ischemia/reperfusion injury by increasing supply of carbon monoxide, biliverdin, or bilirubin). Up-regulation of HO-1 expression has been associated with increased resistance to tissue injury. Bach1 is a bZip protein which forms heterodimers with small Maf proteins. HO-1 is expressed at higher levels in tissues of Bach1-deficient mice, indicating that Bach1 acts as a negative regulator of the mouse HO-1 gene. The molecular mechanism that confers repression of HO-1 by Bach1, and whether there are similar effects in human cells, has remained elusive. The aim of this study was to assess whether modulation of human hepatic Bach1 expression by siRNA technology influences HO-1 gene expression and whether such gene silencing would enhance the inducing effects of heme on HO-1. Methods: siRNAs targeted 4 different positions of human Bach1 mRNA were designed and synthesized. We transfected Bach1-siRNA (25–200 nM) into Huh-7 cells using Lipofectamine for 24–72 h, after which, cells were treated with or without heme. We quantified HO-1 and Bach1 mRNA and protein levels by quantitative RT-PCR and western blotting, respectively. Effects and specificity of Bach1-siRNA were analyzed and compared with those of non-Bach1 related siRNAs (non-specific control-duplex (NSCD) and LaminB2-siRNA). Results: Bach1-siRNAs (25–200 nM) transfected into Huh-7 cells for 24–72 h significantly reduced Bach1 mRNA and protein levels approximately 80%, compared with non siRNA treated cells. In contrast, transfection with same amounts of NSCD or LaminB2 siRNA did not reduce Bach1 mRNA or protein levels, confirming the specificity of Bach1-siRNA in Huh-7 cells. A significant finding of these studies was the 7-fold up-regulation of the HO-1 gene in Bach1-siRNA transfected cells, compared to cells without Bach1-siRNA or those transfected with NSCD or LaminB2. Bach1, NSCD, and LaminB2 siRNAs had no effect on HO-2 or 5-aminolevulinate synthase-1 mRNA levels (two genes that are not induced by heme). The effects of increasing concentrations of heme (up to 10 μM) in the presence or absence of Bach1-siRNA on the levels of HO-1 mRNA expression are shown in the Figure. For all of the heme concentrations tested, the levels of HO-1 mRNA were greater when Bach1 siRNA was present. Conclusions: Bach1 has a specific and selective effect to repress expression of human hepatic HO-1. Silencing of the Bach1 gene by siRNAs may be a useful method for up-regulating HO-1 gene expression. The combination of intravenous heme and Bach1 silencing may be useful for therapy of acute porphyrias in relapse or other conditions in which up-regulation of HO-1 may be beneficial. (Supported by grants from NIH [DK38825] and Ovation Pharmaceuticals, Inc.) Figure Figure