The codon sequences in the designed mRNA vaccine construct were optimized to ensure efficient expression within human cells. For this purpose, the GenSmart Codon Optimization Tool (http://www.genscript.com/ ) provided by GenScript (G.S.) was used. After optimization, a quality assessment of the optimized sequence was performed using the Rare Codon Analysis tools (http://www.genscript.com/ ) also provided by GenScript. The efficiency of mRNA translation was determined using Codon Adaptation Index (CAI). In addition, any unusual tandem codons present in the optimized sequence were identified through codon frequency distribution analysis. By optimizing the codon sequences, the expression and efficacy of the mRNA vaccine construct can potentially be improved by researchers.
Rare codon analysis tool
Manufactured by GenScript
The Rare Codon Analysis Tool is a software application designed to analyze the frequency and distribution of rare codons within a DNA sequence. It provides users with a comprehensive overview of the rare codon usage patterns in their genetic data.
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11 protocols using rare codon analysis tool
1
Codon Optimization for mRNA Vaccine
2
Codon Optimization for Peptide Vaccine
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The peptide vaccine construct needs to undergo a codon optimization for efficient expression within the human cells. Accordingly, we used the GenSmart Codon Optimization Tool (http://www.genscript.com/ ) by GenScript (GS). Quality assessment of the optimized sequence was performed using the Rare Codon Analysis tools (http://www.genscript.com/ ) by GenScript (GS). The efficiency of translation of the mRNA is expressed as Codon Adaptation Index (CAI). The existence of any tandem unusual codons is indicated as Codon Frequency Distribution (CFD).
3
Peptide Vaccine Codon Optimization
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For effective production within human cells, the peptide vaccine construct must be codon optimized. As a result, GenScript’s (GSs) tool of GenSmart Codon Optimization (http://www.genscript.com/ accessed on 10 December 2021) was used. The optimized sequence’s quality was evaluated using GenScript’s Rare Codon Analysis tools. The Codon Adaptation Index indicates the efficiency of mRNA translation (CAI). Codon Frequency Distribution indicated the presence of any tandem uncommon codons (CFD).
4
Optimizing Recombinant Protein Expression in E. coli
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Reverse translation and codon optimization were performed using Codon Usage Wrangler (http://www.mrclmb.cam.ac.uk/ms/methods/codon.html ) and the GenScript Rare Codon Analysis tool (https://www.genscript.com/tools/rare-codon-analysis ), respectively. Codon adaptation index (CAI), GC content, and codon frequency distribution (CFD) were evaluated using the GenScript Rare Codon Analysis. These key parameters influence the protein expression level in the Eschercia coli host. To prepare the adopted sequences for cloning in E. coli, pET-14b vector was chosen and NdeI and BamHI restriction sites were added to the N and C-terminal of the final construct, respectively.
5
Optimized aga-F75m gene for maize expression
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In order to enhance the expression efficiency in transgenic maize, the DNA sequence of aga-F75 from Gibberella sp. F75 (FJ392036) [16 (link)] was modified by removing the putative N-terminal signal peptide-coding sequence, introducing two restriction sites (BamHI and AvrII) at both ends of the gene and optimizing the codons according to the optimal codon usage and known codon bias of maize [23 (link),24 (link)]. Codon adaptation index (CAI) and GC content were analyzed by using the GenScript Rare Codon Analysis Tool (http://www.genscript.com/cgi-bin/tools/rare_codon_analysis ) to assess the modified gene coding sequence and predict the gene expression level. The modified gene, designated aga-F75m, was synthesized by Genscript (Nanjing, China), and cloned into vector pUC57MCS to construct the recombinant plasmid pUC57-aga-F75m.
6
Generating pTagging-iCherry-hyg Vector
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pTagging-iCherry-hyg vector (GenBank MW246024 ) was generated by cloning the hygromycin resistance gene and SV40 polyA fragment [PCR amplified from pTag-OpIE2dsRed (Fernandes et al. 2012 (link))], into NheI digested pTagging-iCherry backbone. The pTagging-iCherry vector (GenScript, GenBank MW246023 ) was in silico designed using Vector NTI and synthesized by GenScript (USA). This cassette is composed by the following elements: the OpIE2 promoter from the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus (OpMNPV) immediate-early 2 (ie-2) gene, FRT wt (Fwt) sequence, icherry gene (a Spodoptera frugiperda codon-optimized version of mCherry designed using the GenScript Rare Codon Analysis Tool) with a polyA from the OpMNPV ie-2 gene, FRT F5 (F5) sequence, and the OpIE1 promoter, all cloned in pUC57.
7
Optimized Can f 6 Gene Synthesis
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Can f 6 has 528 bases pairs, encoding 175 amino acids. Can f 6 gene was optimized on the basis of unchanged amino acid sequences [18 (link)], according to the GenScript rare codon analysis tool (http://www.genscript.com/.cgi-bin/tools/rare_codon_analysis ) and synthesized (GenScript, Nanjing), then subcloned into pET-28a vector with the sites of EcoR I and Xho I and verified by DNA sequencing.
8
Optimizing Triple Fusion Protein Production
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To facilitate triple fusion protein production in plants, the original TF gene sequence was optimized for expression in Nicotiana tabacum by Genscript (Piscataway, NJ, USA) and cloned into the pUC57 vector at the XbaI/SstI restriction sites, giving rise pUC57/TFnt. The Codon Adaptation Index for TF and TFnt genes was calculated using the GenScript Rare Codon Analysis Tool (http://www.genscript.com/cgi-bin/tools/rare_codon_analysis ).
9
Optimizing Mammalian Gene Expression
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A codon-optimized complementary DNA (cDNA) of the two CTL and two
HTL MPVs was generated and analyzed for favored expression in the
mammalian cell line (human) by the Java Codon Adaptation Tool. The
cDNA of MPVs was further analyzed by the GenScript Rare Codon Analysis
Tool for the large-scale expression potential. The tool analyzes the
GC content, codon adaptation index (CAI), and the tandem rare codon
frequency.83 (link),84 (link) The CAI indicates the possibility
of expression in the chosen human cell line expression system. The
tandem rare codon frequency indicates the presence of low-frequency
codons in the cDNA construct.
HTL MPVs was generated and analyzed for favored expression in the
mammalian cell line (human) by the Java Codon Adaptation Tool. The
cDNA of MPVs was further analyzed by the GenScript Rare Codon Analysis
Tool for the large-scale expression potential. The tool analyzes the
GC content, codon adaptation index (CAI), and the tandem rare codon
frequency.83 (link),84 (link) The CAI indicates the possibility
of expression in the chosen human cell line expression system. The
tandem rare codon frequency indicates the presence of low-frequency
codons in the cDNA construct.
10
Codon Optimization and Cloning for Recombinant Protein Expression
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The Java Codon Adaptation Tool (Jcat) (https://www.jcat.de/ ) was utilized for the codon optimization approach to enhance the expression of recombinant protein. Jcat developed a cDNA sequence of the vaccine to do codon optimization and reverse translation at the same time [106 (link)]. To ensure optimal protein expression, GenScript a Rare Codon Analysis Tool (https://www.genscript.com/tools/rarecodonanalysis ) was used to improve the product by analyzing discrete parameters such as G-C concentration and Codon Adaptation Index (CAI) of the sequence [107 (link)]. Finally, cloning procedure was carried out using Snap Gene v4.2 software (https://snapgene.com/ ) by introducing the modified nucleotide sequence into the pET28a (+) expression vector [108 (link)].
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