scholarly journals A Computational Approach to Design Potential siRNA Molecules as a Prospective Tool for Silencing Nucleocapsid Phosphoprotein and Surface Glycoprotein Gene of SARS-CoV-2

2020 ◽  
Author(s):  
Umar Faruq Chowdhury ◽  
Mohammad Umer Sharif Shohan ◽  
Kazi Injamamul Hoque ◽  
Mirza Ashikul Beg ◽  
Mohammad Kawsar Sharif Siam ◽  
...  
Keyword(s):  
Free Energy ◽  
Rna Interference ◽  
Target Genes ◽  
Antiviral Treatment ◽  
Gc Content ◽  
Surface Glycoprotein ◽  
Rnai Pathway ◽  
Viral Gene ◽  
Conserved Sequence ◽  
Glycoprotein Gene

ABSTRACTAn outbreak, caused by a RNA virus, SARS-CoV-2 named COVID-19 has become pandemic with a magnitude which is daunting to all public health institutions in the absence of specific antiviral treatment. Surface glycoprotein and nucleocapsid phosphoprotein are two important proteins of this virus facilitating its entry into host cell and genome replication. Small interfering RNA (siRNA) is a prospective tool of the RNA interference (RNAi) pathway for the control of human viral infections by suppressing viral gene expression through hybridization and neutralization of target complementary mRNA. So, in this study, the power of RNA interference technology was harnessed to develop siRNA molecules against specific target genes namely, nucleocapsid phosphoprotein gene and surface glycoprotein gene. Conserved sequence from 139 SARS-CoV-2 strains from around the globe was collected to construct 78 siRNA that can inactivate nucleocapsid phosphoprotein and surface glycoprotein genes. Finally, based on GC content, free energy of folding, free energy of binding, melting temperature and efficacy prediction process 8 siRNA molecules were selected which are proposed to exerts the best action. These predicted siRNAs should effectively silence the genes of SARS-CoV-2 during siRNA mediated treatment assisting in the response against SARS-CoV-2

scholarly journals An RNA Interference Tool to Silence Genes in Sarcoptes scabiei Eggs

2022 ◽  
Vol 23 (2) ◽  
pp. 873
Author(s):  
Deepani D. Fernando ◽  
Pasi K. Korhonen ◽  
Robin B. Gasser ◽  
Katja Fischer
Keyword(s):  
Rna Interference ◽  
Drug Targets ◽  
Target Genes ◽  
Developmental Stages ◽  
Single Copy ◽  
Sarcoptes Scabiei ◽  
Rnai Pathway ◽  
Larval Hatching ◽  
Parasitic Mite ◽  
Pre Treatment

In a quest for new interventions against scabies—a highly significant skin disease of mammals, caused by a parasitic mite Sarcoptes scabiei—we are focusing on finding new intervention targets. RNA interference (RNAi) could be an efficient functional genomics approach to identify such targets. The RNAi pathway is present in S. scabiei and operational in the female adult mite, but other developmental stages have not been assessed. Identifying potential intervention targets in the egg stage is particularly important because current treatments do not kill this latter stage. Here, we established an RNAi tool to silence single-copy genes in S. scabiei eggs. Using sodium hypochlorite pre-treatment, we succeeded in rendering the eggshell permeable to dsRNA without affecting larval hatching. We optimised the treatment of eggs with gene-specific dsRNAs to three single-copy target genes (designated Ss-Cof, Ss-Ddp, and Ss-Nan) which significantly and repeatedly suppressed transcription by ~66.6%, 74.3%, and 84.1%, respectively. Although no phenotypic alterations were detected in dsRNA-treated eggs for Ss-Cof and Ss-Nan, the silencing of Ss-Ddp resulted in a 38% reduction of larval hatching. This RNAi method is expected to provide a useful tool for larger-scale functional genomic investigations for the identification of essential genes as potential drug targets.

scholarly journals Assessment of siRNA as a therapeutic molecule in Transient Receptor Potential Channel 5 gene silencing: a computational approach

2018 ◽  
Vol 5 (1) ◽  
pp. 1911-1922
Author(s):  
Bhooma Vijayaraghavan ◽  
Giri Padmanabhan ◽  
Kumaresan Ramanathan
Keyword(s):  
Free Energy ◽  
Gene Silencing ◽  
Target Genes ◽  
Transient Receptor Potential ◽  
Gc Content ◽  
Receptor Potential ◽  
Transient Receptor Potential Channel ◽  
Minimum Free Energy ◽  
Transient Receptor

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.

scholarly journals Prediction and in vitro silencing of two lipid biosynthetic genes viz. SCD and SREBP1 in chicken using RNA interference

2020 ◽  
Author(s):  
N. Govardhana Sagar ◽  
A. Rajendra Prasad ◽  
R.N. Chatterjee ◽  
Pushpendra Kumar ◽  
Bharat Bhushan ◽  
...  
Keyword(s):  
Cell Culture ◽  
Rna Interference ◽  
Culture System ◽  
Target Gene ◽  
Target Genes ◽  
Gc Content ◽  
Transcriptional Silencing ◽  
Cell Culture System ◽  
Knock Down

AbstractRNA interference is a widely used post transcriptional silencing mechanism for suppressing expression of the target gene. In the current study, five shRNA molecules each against SCD and SREBP1 genes were designed after considering the parameters like secondary structures of shRNA constructs, mRNA target regions, GC content and thermodynamic properties (ΔG overall, ΔG duplex and ΔG break-target) to knockdown their expression. After successful transfection of these shRNA molecules into the chicken embryonic hepatocyte culture, expression of the target genes were monitored by real time PCR. Significant reduction (P<0.05) in the expression of SCD and SREBP1 genes with minimal activation of immune response genes in hepatocytes was observed after transfecting the shRNA molecules into them. The shRNA constructs against SCD gene showed the knock down efficiency ranged from 20.4% to 74.2%. In case of shRNA constructs against SREBP1 gene, they showed knock down efficiency ranging from 26.8% to 95.85%. These results clearly demonstrated the successful down regulation of the gene expression by designed shRNA molecules against both the target genes under in vitro condition. The shRNA2 molecule for SCD gene and the shRNA1 molecule for SREBP1 gene were found as the best among all the shRNA molecules used for silencing the target genes under cell culture system. It is concluded that the shRNA molecules designed against SCD and SREBP1 genes showed potential to silence the expression of these genes under in vitro chicken embryonic hepatocyte cell culture system.

scholarly journals RNA Interference of Genes Encoding the Vacuolar-ATPase in Liriomyza trifolii

Insects ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 41
Author(s):  
Ya-Wen Chang ◽  
Yu-Cheng Wang ◽  
Xiao-Xiang Zhang ◽  
Junaid Iqbal ◽  
Yu-Zhou Du
Keyword(s):  
Rna Interference ◽  
Expression Analysis ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Control Strategies ◽  
Vacuolar Atpase ◽  
Invasive Pest ◽  
Liriomyza Trifolii ◽  
Horticultural Crops ◽  
Genes Encoding

The leafminer fly, Liriomyza trifolii, is an invasive pest of vegetable and horticultural crops in China. In this study, a microinjection method based on dsRNA was developed for RNA interference (RNAi) in L. trifolii using genes encoding vacuolar-ATPase (V-ATPase). Expression analysis indicated that V-ATPase B and V-ATPase D were more highly expressed in L. trifolii adults than in larvae or pupae. Microinjection experiments with dsV-ATPase B and dsV-ATPase D were conducted to evaluate the efficacy of RNAi in L. trifolii adults. Expression analysis indicated that microinjection with 100 ng dsV-ATPase B or dsV-ATPase led to a significant reduction in V-ATPase transcripts as compared to that of the dsGFP control (dsRNA specific to green fluorescent protein). Furthermore, lower dsRNA concentrations were also effective in reducing the expression of target genes when delivered by microinjection. Mortality was significantly higher in dsV-ATPase B- and dsV-ATPase D-treated insects than in controls injected with dsGFP. The successful deployment of RNAi in L. trifolii will facilitate functional analyses of vital genes in this economically-important pest and may ultimately result in new control strategies.

A bioinformatics approach to investigate serum and hematopoietic cell-specific therapeutic microRNAs targeting the 3' UTRs of all four Dengue virus serotypes

2021 ◽  
Author(s):  
Mirza Sarwar Baig ◽  
Anuja Krishnan
Keyword(s):  
Dengue Virus ◽  
Antigen Processing ◽  
Target Genes ◽  
Public Health Problem ◽  
Hematopoietic Cell ◽  
Viral Gene ◽  
P Value ◽  
Pathway Enrichment Analysis ◽  
Global Public Health ◽  
Denv Serotypes

Abstract Hyperendemic circulation of all four Dengue virus (DENV) serotypes is a severe global public health problem, so any vaccine or therapeutics should be able to target all four of them. Cells of hemopoietic origin are believed to be primary sites of DENV replication. This study aimed to identify potential host miRNAs that target 3' UTR of all 4 DENV serotypes, thereby directly regulating viral gene expression or indirectly modulating the host system at different virus infection steps. We used four prediction algorithms viz. miRanda, RNA22, RNAhybrid, and StarMir for predicting miRNA, targeting 3'UTR of all four DENV serotypes. Statistically, the most significant miRNA targets were screened based on their Log10 P-value (&gt; 0.0001) of GO term and KEGG pathway enrichment analysis. The intersection test of at least three prediction tools identified a total of 30 miRNAs, which could bind to 3'UTR of all four DENV serotypes. Of the 30, eight miRNAs were of hematopoietic cell origin. GO term enrichment and KEGG analysis showed four hemopoietic origin miRNAs target genes of the biological processes mainly involved in the innate immune response, mRNA 3'-end processing, antigen processing, and presentation and nuclear-transcribed mRNA catabolic process.

A strand bias occurs in point mutations associated with variant surface glycoprotein gene conversion in Trypanosoma rhodesiense

1994 ◽  
Vol 14 (6) ◽  
pp. 3971-3980
Author(s):  
Y Lu ◽  
C M Alarcon ◽  
T Hall ◽  
L V Reddy ◽  
J E Donelson
Keyword(s):  
Point Mutations ◽  
Cross Reactivity ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Strand Bias ◽  
Glycoprotein Gene ◽  
Coding Regions ◽  
Crossover Site ◽  
Sense Strand ◽  
Duplication Events

We previously described a bloodstream Trypansoma rhodesiense clone, MVAT5-Rx2, whose isolation was based on its cross-reactivity with a monoclonal antibody (MAb) directed against a metacyclic variant surface glycoprotein (VSG). When the duplicated, expressed VSG gene in MVAT5-Rx2 was compared with its donor (basic copy) gene, 11 nucleotide differences were found in the respective 1.5-kb coding regions (Y. Lu, T. Hall, L. S. Gay, and J. E. Donelson, Cell 72:397-406, 1993). Here we describe a characterization of two additional bloodstream trypanosome clones, MVAT5-Rx1 and MVAT5-Rx3, whose VSGs are expressed from duplicated copies of the same donor VSG gene. The three trypanosome clones each react with the MVAT5-specific MAb, but they have different cross-reactivities with a panel of other MAbs, suggesting that their surface epitopes are similar but nonidentical. Each of the three gene duplication events occurs at a different 5' crossover site within a 76-bp repeat and is associated with a different set of point mutations. The 35, 11, and 28 point mutations in the duplicated VSG coding regions of Rx1, Rx2, and Rx3, respectively, exhibit a strand bias. In the sense strand, of the 74 total mutations generated in the three duplications, 54% are A-to-G or G-to-A (A:G) transitions and 7% are C:T transitions, while 26% are C:A transversions and 13% are C:G transversions. No T:G or T:A transversions occurred. Possible models for the generation of these point mutations are discussed.

scholarly journals Understanding the Use of Real Time Reverse Transcriptase-Polymerase Chain Reaction (rRT-PCR) For Covid-19 Diagnosis

2020 ◽  
Vol 4 (2) ◽  
pp. 86-97
Author(s):  
Adesola O. Olalekan ◽  
Bamidele A. Iwalokun ◽  
Olutoyin C. Adekunle ◽  
Hussaini A. Makun ◽  
Tatfeng Mirabeau ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Target Genes ◽  
Protein Gene ◽  
Rna Detection ◽  
Glycoprotein Gene ◽  
Adequate Knowledge ◽  
Alternative Testing ◽  
Polymerase Chain ◽  
Testing Protocols

Background: Adequate knowledge of real time Reverse Transcriptase-Polymerase Chain Re-action (rRT-PCR) is critical for accurate implementation of the assay, interpretation of results and report-ing. This mini-review describes the principles, procedures, and level of development of rRT-PCR assays for the control of the COVID-19 pandemic. Methods: A narrative review was carried out to describe the principles of rRT-PCR, provide an update on the landscape of rRT-PCR protocols and elucidate the process control involved in pre-analytical, analytical and post-analytical stages of COVID-19 testing . Review Findings: The rRT-PCR is currently considered to be the acceptable standard for confirming COVID-19 diagnosis based on SARS-CoV-2 RNA detection via conversion to cDNA and amplification of target genes in real time using sequence specific TaqMan® probes. Available evidence indicates that different rRT-PCR protocols varying in number and type of target genes within SARS-CoV-2 genome are currently available for validation and emergency use approval (EUA) in pandemic countries. A total of 1 – 3 target genes, comprising the ORF1a, ORF1b, RNA dependent RNA polymerase (RdRp), Nucleoplasid protein gene (N), Spike glycoprotein gene (S) and Envelope protein gene (E) are detected by these proto-cols. Conclusion: rRT-PCR remains the most sensitive method for confirming, monitoring and managing COVID-19 disease in the ongoing pandemic in all affected countries. The need for validation of every rRT-PCR protocol prior to deployment for COVID-19 testing and research into the development of alternative testing protocols are strongly recommended

DNA rearrangements and antigenic variation in Trypanosoma equiperdum: expression-independent DNA rearrangements in the basic copy of a variant surface glycoprotein gene

1983 ◽  
Vol 3 (3) ◽  
pp. 410-414
Author(s):  
S Longacre ◽  
A Raibaud ◽  
U Hibner ◽  
G Buck ◽  
H Eisen ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Dna Rearrangements ◽  
Present Evidence ◽  
Glycoprotein Gene ◽  
Copy Gene ◽  
Genomic Environment ◽  
Trypanosoma Equiperdum

Antigenic variation in Trypanosoma equiperdum is associated with the sequential expression of variant surface glycoprotein (VSG) genes in a process which involves gene duplication and transposition events. In this paper we present evidence that the genomic environment of the VSG-1 basic copy gene, the template for duplicated, expression-linked VSG-1 genes, differs in every trypanosome clone examined. This variation is thus independent of the expression of the VSG-1 gene, and it also appears to be restricted to the 3' genomic environment. It is also demonstrated that the DNA located 3' to the VSG-1 basic copy gene is moderately sensitive to digestion when the nuclei of either expressor or non-expressor trypanosomes are treated with DNase I.

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