key: cord-1007920-idfrcykd authors: Bappy, Syed Shahariar; Shibly, Abu Zaffar; Sultana, Sorna; Mohiuddin, A.K.M.; Kabir, Yearul title: Designing Potential siRNA Molecule for the Nucleocapsid(N) Gene Silencing of Different SARS-CoV-2 Strains of Bangladesh: Computational Approach date: 2021-05-06 journal: Comput Biol Chem DOI: 10.1016/j.compbiolchem.2021.107486 sha: b1ce39b4bde13b3939c205bf6760435f103ba773 doc_id: 1007920 cord_uid: idfrcykd SARS-CoV-2 is a single-stranded RNA (+) virus first identified in China and then became an ongoing global outbreak. In most cases, it is fatal in humans due to respiratory malfunction. Extensive researches are going to find an effective therapeutic technique for the treatment of SARS-CoV-2 infected individuals. In this study, we attempted to design a siRNA molecule to silence the most suitable nucleocapsid(N) gene of SARS-CoV-2, which play a major role during viral pathogenesis, replication, encapsidation and RNA packaging. At first, 270 complete N gene sequences of different strains in Bangladesh of these viruses were retrieved from the NCBI database. Different computational methods were used to design siRNA molecules. A siRNA molecule was built against these strains using the SiDirect 2.0 server. Using Mfold and the OligoCalc server, the siRNA molecule was tested for its secondary structure and GC material. The Clustal Omega tool was employed to evaluate any off-target harmony of the planned siRNA molecule. Herein, we proposed a duplex siRNA molecule that does not fit any off-target sequences for the gene silencing of SARS-CoV-2. To treat SARS-CoV-2 infections, currently, any effective therapy is not available. Our engineered siRNA molecule could give an alternative therapeutic approach against various sequenced SARS-CoV-2 strains in Bangladesh. A deadly bat-borne Coronaviridae family virus SARS-CoV-2 was identified first in mid-December of 2019 in Wuhan, China (1) . Since then, it has become an ongoing outbreak worldwide. To this date, it has affected 213 countries and territories across the globe (2) . In this positive sense, the single-stranded RNA virus has already infected about thirteen million people worldwide till July 2020 (3). Patients suffering from SARS-CoV-2 infection are usually found with complications in the respiratory tract, diarrhea, high fever, thrombocytopenia, lymphopenia, and increased C-reactive protein and lactate dehydrogenase levels within the first 3-6 days of viral exposure (1, 4) . With a genome size of ~30000 bases, one of the largest nonsegmented genomes among all RNA viruses, this virus has already compiled plentiful changes in its genome and procures more (5, 6) . Due to the sequencing technologies advancement, various databases preserves the available whole genome sequence of these deadly virus strains now. Researchers are looking for effective molecular therapy against the SARS-CoV-2 using these resources. Bangladeshi scientists also sequenced different SARS-CoV-2 strains, where 273 sequences were available in the NCBI public database until December 2020 (7, 8) . A comparative analysis computed the origin of the various strains of this virus in Bangladesh among publicly available SARS-CoV-2 genome sequences from 27 countries. Their prediction about Bangladeshi strains based on phylogenetic analysis reported that the pathogen appeared from multiple countries (9) . This study was on 64 SARS-CoV-2 isolated genomic sequences from Bangladesh with the reference (NC_045512.2 first Chinese SARS-CoV-2 sequence) and 433 sequences of the whole world available from several countries. The sequences of different Bangladeshi strains were showed three clusters, among them 43 of the 64 sequences shared a comparable node with J o u r n a l P r e -p r o o f Germany, which carried a common ancestor with the United Kingdom. Two other clusters of Bangladesh had 4 and 5 sequences, respectively, and in both cases, they shared the same node with the sequence of SARS-CoV-2 reported from India and Saudi Arabia. On the other hand, 12 sequences that did not belong to any clusters were found to be similar with sequences from Europe, including United Kingdom, Germany, France, Italy, and Russia, while one of these sequences was close to the sequence reported from the USA (9, 10). The underlying link of Bangladeshi SARS-CoV-2 isolates with part of a haplotype observed high in Europe (9, 11) . Though the virus is a mutated particle over time, the SARS-CoV-2 sequence shares 79.6% similarity to SARS-CoV (12, 13) , with the fewer mutations and higher (90%) homology showing N gene, which is more stable and conserved over time, but the S gene has 76% similarity (13) (14) (15) (16) (17) (18) . Nucleocapsid gene silencing Small Interfering RNA (siRNA) molecules potentiality has already been reported (19) . Double-stranded 20-25 base pairs containing RNA generally work through the RNA interference (RNAi) pathway to silence a specific gene expression, where it causes mRNA breakage into the following transcription (20) . These mRNA degrading small molecules play an important role in the post-transcriptional gene silencing (PTGS) (21) . In-vitro studies have verified that siRNA can significantly repress gene expression in mammalian cells (22) (23) (24) . In addition, effective in vivo silencing of the endogenous gene and transgene expression is already revealed (25) (26) (27) (28) . The capability of small interfering RNA is already proved in a in vitro study through the inhibition of SARS-CoV N gene expression in cultured cells and mouse muscles (29) . More than 80% blocking activity was shown utilizing chemically synthesized siRNA duplexes targeting genomic RNA of SARS-CoV (30) . Like other viruses, SARS-CoV-2 nucleocapsid(N) protein is a multifunctional protein and plays a crucial role in encapsidation, viral RNA transcription, and replication (31) . For this reason, the study of the nucleocapsid protein or N gene has become much popular as a diagnostic and therapeutic target (32, 33) . However, some studies show a mutation in the N gene, which is not the focus of this study. Moreover, our main objective is to identify a common therapeutic target for different SARS-CoV-2 strains of Bangladesh. Targeting this sequence may cause SARS-CoV-2 viral inhibition like SARS-CoV by impeding the N gene's translation and thus preventing the RNA transcription and replication (23, 34) . The complete workflow is summarized into the methodology (Figure 1) The selection of SARS-CoV-2 strains and their associated information, including their genus, family, host, transmission pattern, disease pathogenicity, genome, proteome, and the available therapeutic agents against them, was identified ViralZone (http://viralzone.expasy.org/) of the Ex-PASy Bioinformatics Resource Portal. Two hundred and seventy-three sequences of different SARS-CoV-2 (BDG) strains were collected from the viral gene bank database available at NCBI (http://www.ncbi.nlm.nih.gov/). Among them, 270 complete cds of nucleocapsid(N) protein gene was selected, and the other 3 incomplete sequences were disqualified for the next experiment. Multiple sequence alignment of the retrieved sequences was done by Clustal Omega (35) (https://www.ebi.ac.uk/Tools/msa/clustalo/), and phylogeny was analyzed by Phylogeny analysis (http://www.phylogeny.fr/simple_phylogeny.cgi) tool. A web server siDirect 2.0 (http://siDirect2.RNAi.jp/) (36) was used for efficient and targetspecific siRNA design for mammalian RNAi. It utilized Ui-Tei, Amarzguioui, and Reynolds rules combined (37, 38, 39) , and as a parameter, melting temperature (Tm) below 21.5°C for siRNA duplex was also used. Besides, these other parameters were taken on the concept of algorithms given in Table 1 . Blast tool(41) (http://www.ncbi.nlm.nih.gov/blast) was used to identify off-target similarity with any sequence on whole Gene bank datasets other than the target sequence by applying expected threshold value 10 as a parameter. Oligonucleotide Properties Calculator for GC content calculation of predicted siRNA, OligoCalc To study the thermodynamics of interaction between predicted siRNA and target gene Finally, the siRNAPred (45) (http://imtech.res.in/raghava) server from Imtech was used to validate the predicted siRNA further. Here we used efficacy prediction for 21 mers. The predicted siRNA was screened against the Main 21 dataset using a binary pattern. siRNAPred score greater than 0.9 predicts very high efficacy, a score ranging 0.8-0.9 indicates high efficacy, and a score ranging 0.7-0.8 predicts moderate efficacy. At Designing a fruitful siRNA molecule to target a particular gene has a number of challenges. Finding an operative delivery manner is the main challenge; further challenges are off-target silencing, the creation of the immune reaction, and finally, the siRNA stability (49) . Thus, scientists have improved some models to prophesy the efficacy for a deliberate siRNA fragment before in vivo investigation-named Ui-Tei rules, Amarzguioui rules, and Reynolds rule (50) . In this study, we also follow all of these rules to designing a siRNA molecule. The proposed siRNA is covered the threshold score 6 of Reynolds rules, which indicates a proficient siRNA. The GC substance was also found to be within the supported range of 30-52% of the proposed siRNA (56) in vivo, which represents strong protection against these lethal viruses. In recent times, application of polyethyleneimine has been extended towards the complexation and delivery of RNA molecules, especially siRNAs (57) . While chemically unchanged RNA molecules are very unstable with having high degrading possibility, but the enzymatic or nonenzymatic degradation is completely protected by PEI complexation. Before or after influenza virus infection, PEI promoted siRNAs' delivery into the lungs through IV route decrease virus production (58) . In vitro activity also proved for siRNA nanoplexes which prevents siRNAs from serum devastation (59) . Polyethyleneimine and siRNA complexes are effectively delivered into particular cells in vitro, specifically low molecular weight PEI exhibit high siRNA protection and delivery efficacies (60) . Lyophilization preserves physical stability and biological activity of the PEI/RNA (siRNA or ribozyme) complexes under some conditions (60, 61) . J o u r n a l P r e -p r o o f Molecular therapy is replacing conventional therapeutic approaches as a promising alternative way because of its specificity and successive nature. Potentiality of siRNAs have already afford to effectual cure for different diseases. In this study, we have anticipated that a siRNA molecule to apply as therapeutic agent to combat SARS-CoV-2. Functionality and stability of the predicted siRNA molecule is also defined using various algorithms with their suitable parameters. Molecular therapies are precise to DNA sequences that are sometimes nonfunctional against different strains. Our designed siRNA is expected to overcome this issue as it has targeted a conserved sequence found in different SARS-CoV-2 strains, which Bangladeshi scientists sequence. 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