key: cord-0812353-exoc6xvt authors: Chowdhury, Umar Faruq; Shohan, Mohammad Umer Sharif; Hoque, Kazi Injamamul; Beg, Mirza Ashikul; Siam, Mohammad Kawsar Sharif; Moni, Mohammad Ali title: A Computational Approach to Design Potential siRNA Molecules as a Prospective Tool for Silencing Nucleocapsid Phosphoprotein and Surface Glycoprotein Gene of SARS-CoV-2 date: 2020-04-21 journal: bioRxiv DOI: 10.1101/2020.04.10.036335 sha: 94afe01b2f88726280f472ac0871d09c3634c978 doc_id: 812353 cord_uid: exoc6xvt An 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 COVID-19, A pandemic affecting lives of billions of people worldwide, has confronted humanity in 38 the commencement of 2020, is caused by a viral pathogen, severe acute respiratory syndrome 39 coronavirus 2 (SARS-CoV-2) or 2019-nCoV. Initial symptoms of this disease mainly include fever, 40 cough, fatigue, dyspnea & headache [1, 2] or it may be asymptomatic [3] . The spike glycoprotein of 41 SARS-CoV-2 binds directly with the surface cell angiotensin converting enzyme II (ACE2) receptor 42 present on alveolar epithelial cells of lung facilitating virus entry, replication and triggers cytokine 43 cascade mechanism [4] . In severe cases, patient may die due to massive alveolar damage and 44 progressive respiratory failure [1, 5] . The current detection process of SARS-CoV-2 carried out by 45 most countries is using real-time RT-PCR, although several other methods are also being 46 developed [6] [7] [8] . Incubation period for the virus ranges between 2-14 days [9] and in some cases, 47 transmission is also reported during asymptomatic period [10] . Some recent studies suggest that bats 48 are likely reservoir hosts for SARS-Cov-2 but the identity of the intermediate host that might have 49 facilitated transfer to human still remain elusive with some studies indicating pangolins [11] . 50 SARS-CoV-2 is assumed to spread mainly from person-to-person through respiratory droplets 51 produced when an infected person sneezes and coughs or between people who are in close 52 contact [5] . 53 54 Coronaviruses are genetically classified into four main genera: Alphacoronavirus, Betacoronavirus, 55 Gammacoronavirus, and Deltacoronavirus [12] . The first two genera generally infect mammals, 56 while the last two mostly cause disease in birds. The genome size of coronaviruses ranges between 57 approximately 26-32 kb and includes about 6 to 11 open reading frames (ORFs) [13] . Nucleocapsid 58 protein (N), small envelope protein (E), spike surface glycoprotein (S) and matrix protein (M) are 59 the four major structural proteins of coronavirus and all of which are essential to produce a 60 structurally complete virus [14, 15] CoV-2 from around the world. Phylogenetic analysis revealed that a small number sequences form 235 significant clades with a bootstrap value greater than 60%. (Fig 2, Supplementary Table 8 ). 236 Conserved portions that are shorter than 21 nucleotides were omitted from further analysis. 237 Conserved sequences were put to siDirect web server to identify possible targets and to generate 238 corresponding siRNAs. siDirect performs the task in three distinct steps -highly functional siRNA 239 selection, seed-dependent off-target effects reduction, near-perfect matched genes elimination. 240 siRNA targets were found in 18 conserved regions, 5 nucleocapsid phosphoprotein (Supplementary 241 Table 4 ) and 13 surface glycoprotein (Supplementary Table 5 the prediction of prospective secondary structure and determination of free energy of corresponding 264 folding is crucial. Here, guide strands of predicted siRNAs were subjected to RNA structure web 265 server in order to predict possible folding structures and corresponding minimum free energies. At 266 37•C, finally selected siRNAs have free energy of folding greater than zero (Fig 3, Table 2 ), which 267 suggests the predicted siRNAs are more accessible for efficient binding. Table 6 & Supplementary table 7) . Finally selected 273 siRNAs have free energy of binding equal or below -30.0 (Fig 4, Table 2 ), which suggests the 274 predicted siRNAs are more interactive with their corresponding targets. 275 276 The collective heat capacity, denoted as Cp, is plotted as a function of temperature and the melting 277 temperature, denoted as Tm (Cp), was determined. In this study, eight prospective siRNA molecules were proposed to be efficient at binding and 290 cleaving specific mRNA targets of SARS-CoV-2 ( into the endosomes. siRNA escape endosomes and release siRNA into the cytoplasm due to pH 510 responsive mechanism or proton sponge effect. Once generated, siRNA is loaded into RNA-indiced 511 silencing complex comprising of RNA-binding protein TRBP and Argonaute (AGO2). AGO2 opts 512 the siRNA guide strand, then excises and ejects the passenger strand. After that, the guide strand 513 pairs with its complementary target mRNA and AGO2 slices the target. After slicing, the cleaved 514 target mRNA is released and RISC is recycled for another few rounds of slicing using the same 515 guide Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus 350 Incubation Period and Other Epidemiological Characteristics of Novel Coronavirus Infections with Right Truncation: A Statistical Analysis of Publicly 355 Available Case Data A Familial Cluster of Infection Associated With the Novel Coronavirus Indicating Possible Person-to-Person Transmission During the Incubation 358 Period CoV-2 related coronaviruses in Malayan pangolins Structure, Function, and Evolution of Coronavirus Spike Proteins From 366 SARS to MERS, thrusting coronaviruses into the spotlight The Molecular Biology of Coronaviruses MERS-CoV virus-like particles produced in insect cells 372 induce specific humoural and cellular imminity in rhesus macaques Structure of the N-terminal RNA-binding domain of the SARS CoV 376 nucleocapsid protein Molecular Interactions in the Assembly of Coronaviruses Mass spectroscopic characterization of the coronavirus infectious bronchitis virus 382 nucleoprotein and elucidation of the role of phosphorylation in RNA binding by using 383 surface plasmon resonance An interaction between the 386 nucleocapsid protein and a component of the replicase-transcriptase complex is crucial for 387 the infectivity of coronavirus genomic RNA Severe 390 acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and 391 nucleocapsid proteins function as interferon antagonists Structural insights into coronavirus entry Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory 397 Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus 398 Entry and Cell-Cell Fusion Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer, 402 Proteolytic 404 processing of middle east respiratory syndrome coronavirus spikes expands virus tropism RNA interference as a prospective tool for the control of 408 human viral infections Computational design of potential siRNA 411 molecules for silencing nucleoprotein gene of rabies virus Against Leishmania donovani by Predicting RNAi Molecules Against the Surface Protein, 415 gp63 A species of small antisense RNA in posttranscriptional 418 gene silencing in plants, Science (80-. ) Prophylactic and therapeutic effects of small 422 interfering RNA targeting SARS-coronavirus Scaffold of N-(2-(2-424 (tosylcarbamoyl)hydrazinyl)ethyl)isonicotinamidereveals anticancer effects through selective 425 inhibition of FAP Molecular docking reveals 428 pitavastatin and related molecules antagonize 1DHF and its pseudogene DHFR2 in cancer 429 treatment Drug repurposing: Targeting 432 mTOR inhibitors for anticancer activity In Silico structure based designing of 436 dihydrofolate reductase enzyme antagonists and potential small molecules that target DHFR 437 protein to inhibit the folic acid biosynthetic pathways Surface proteins, potential drug 441 target for antiviral therapy against Nipah virus and in silico drug design The Nucleoprotein Is Required for Efficient Coronavirus 444 Inhibition of SARS-CoV replication 447 by siRNA Virus Variation Resource -improved response to emergent viral 450 outbreaks Improving the sensitivity of 452 progressive multiple sequence alignment through sequence weighting, position-specific gap 453 penalties and weight matrix choice Estimation of the number of nucleotide substitutions in the control region 456 of mitochondrial DNA in humans and chimpanzees Molecular evolutionary 459 genetics analysis across computing platforms MEGA-CC: computing core of molecular 462 evolutionary genetics analysis program for automated and iterative data analysis Interactive Tree Of Life (iTOL) v4: recent updates and new 465 developments SiDirect 2.0: Updated software for designing 468 functional siRNA with reduced seed-dependent off-target effect Guidelines for the selection of highly effective siRNA sequences for mammalian and 472 chick RNA interference An algorithm for selection of functional siRNA sequences Rational siRNA 478 design for RNA interference 481 BLAST+: Architecture and applications OligoCalc: an online oligonucleotide properties calculator Improved RNA secondary structure prediction by 486 maximizing expected pair accuracy RNAstructure: web servers for RNA 489 secondary structure prediction and analysis A sequence similar to 492 tRNA3Lys gene is embedded in HIV-1 U3-R and promotes minus-strand transfer DINAMelt web server for nucleic acid melting prediction siRNApred: SVM based method for 497 predicting efficacy value of siRNA A 499 structural interpretation of the effect of GC-content on efficiency of RNA interference, BMC 500 Bioinforma A three-dimensional view of the molecular machinery of RNA 502