<p><h3><i>In silico</i> design of Influenza Toehold switches</h3></p>
<p><h3><i>In silico</i> design of Influenza Toehold switches</h3></p>
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According to Green et al., the optimal length of RNA to be detected by a toehold switch is around 30 bp. In other words, a target RNA with 1000 bp in length will give 970 possible switches. However, the performances of each possible switch are different, since the performance is governed by serval parameters in the target region, such as the minimum free energy (For more information, please visit <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model">RNA thermodynamics modelling page</a>). To minimize the manpower on screening of the switches, we constructed an <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Software"> online toehold switch design program </a>. Apart from basic thermodynamic parameters, it also screens for rare codon, stop codon and RFC illegal sites along the sequence. In addition, the built- in BLAST function also automatically screen for nonspecific region to avoid false positive detection. Ultimately, the program can sort a list of “best” Toehold Switch sequence according to their free energy using the embedded function of <a href="https://www.tbi.univie.ac.at/RNA/">“Vienna RNA”</a> (8). The program facilitates the construction of toehold switch by providing a user-friendly interface with novel screening function.
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According to Green <i>et al.</i>, the optimal length of RNA to be detected by a toehold switch is around 30 bp. In other words, a target RNA with 1000 bp in length will give 970 possible switches. However, the performances of each possible switches are different, since the performance is governed by serval parameters in the target region, such as the minimum free energy of the RNA (For more information, please visit <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model">RNA thermodynamics modelling page</a>). To minimize the manpower on screening of the switches, we constructed an <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Software"> online toehold switch design program </a>. Apart from the basic thermodynamic parameters, it also screens for rare codons, stop codons and RFC illegal sites along the sequence. In addition, the built-in BLAST function also automatically screen for nonspecific region to avoid false positive detection. Ultimately, the program can sort a list of “best” Toehold Switch sequence according to their free energy using the embedded function of <a href="https://www.tbi.univie.ac.at/RNA/">“Vienna RNA”</a> (8). The program facilitates the construction of toehold switch by providing a user-friendly interface with novel screening function.
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We used the program to design our switches to detect H5, H7, N1, N9 and PB2 RNAs. Polymerase basic protein 2 (PB2) gene is influenza A specific gene that used as a positive control of flu A. We downloaded the latest influenza gene sequence from the Influenza Research Database and inputted to our program to generate switches. The sequence used are listed below:
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To detect influenza A, Polymerase Basic Protein 2 (PB2) gene is used as a positive control as it is influenza A-specific. Further subtyping requires a subtype-specific RNA that can also fulfil the criteria for being a good toehold switch. We downloaded the latest influenza gene sequences from the Influenza Research Database and inputted to our program to generate switches to detect H5, H7, N1, N9 and PB2 RNAs. The sequences used are listed below:
We chose 3 toehold switches with “good” predicted performance to target each RNA (For more information, please visit <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model">RNA thermodynamics modelling page</a>). (e.g. H5-1, H5-2 and H5-3 to detect H5 RNA). The figure above shows the detection region of each toehold switch. Before constructing the toehold switches, we ensured all the switches passed the modelling test.
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We chose 3 toehold switches with “good” predicted performance to target each RNA (For more information, please visit <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model">RNA thermodynamics modelling page</a>). (e.g. H5-1, H5-2 and H5-3 to detect H5 RNA). The figure above shows the detection region of each toehold switch. Before constructing the toehold switches, we ensured all the switches passed our modelling criteria.