Difference between revisions of "Part:BBa K5106001"
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<partinfo>BBa_K5106001 short</partinfo> | <partinfo>BBa_K5106001 short</partinfo> | ||
| − | This toehold switch is designed for detection of hsa- | + | This toehold switch is designed for detection of hsa-miR-484 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106000 BBa_K5106000]</span>), a miRNA which is shown to be present at increased levels in the presence of multiple sclerosis. This is the first of two toehold switches, designed with an improved version of the SwitchMi design model (UParis BME iGEM team 2021), modified by <span class="plainlinks">[https://2024.igem.wiki/wageningenur miRADAR]</span> (WageningenUR 2024). |
==Introduction== | ==Introduction== | ||
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<br> | <br> | ||
</html> | </html> | ||
| − | <small>'''Figure 1:''' a) General structure of a toehold switch RNA, | + | <small>'''Figure 1:''' a) General structure of a toehold switch RNA, consisting of a complementary toehold domain, a hairpin structure formed by the ribosome binding site and start codon, and a repressed gene. b) Simplified mechanism of a toehold switch. The trigger (mi)RNA, which is complementary to the toehold region, binds to the switch RNA (depicted in yellow), allowing the hairpin to linearize. This allows for binding of the ribosome and translation of the repressed mRNA</small> |
When the trigger RNA is not present, the RBS is in the hairpin structure, disabling the possibility for the ribosome to bind, and thereby preventing translation of the protein. However, when the trigger RNA is present, it can bind the toehold domain of the switch, thereby opening the hairpin structure by a strand displacement reaction. This exposes the RBS and start codon, allowing ribosome binding and subsequent mRNA translation (Figure 1b). | When the trigger RNA is not present, the RBS is in the hairpin structure, disabling the possibility for the ribosome to bind, and thereby preventing translation of the protein. However, when the trigger RNA is present, it can bind the toehold domain of the switch, thereby opening the hairpin structure by a strand displacement reaction. This exposes the RBS and start codon, allowing ribosome binding and subsequent mRNA translation (Figure 1b). | ||
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This part codes for a toehold switch that contains a region that is complementary to the microRNA hsa-miR-484 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106000 BBa_K5106000]</span>), which is upregulated in the presence of multiple sclerosis. In the presence of this miRNA, this trigger can bind to the toehold switch RNA, allowing for translation of a downstream protein coding region. This part can regulate expression of any protein in response to the presence of hsa-miR-484. | This part codes for a toehold switch that contains a region that is complementary to the microRNA hsa-miR-484 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106000 BBa_K5106000]</span>), which is upregulated in the presence of multiple sclerosis. In the presence of this miRNA, this trigger can bind to the toehold switch RNA, allowing for translation of a downstream protein coding region. This part can regulate expression of any protein in response to the presence of hsa-miR-484. | ||
| − | This part was designed using the tool designed by miRADAR for designing new toehold switches for miRNA detection, which is a modified version of the SwitchMi design model (UParis BME iGEM team 2021). The conserved regions and the structure of the toehold switch are based on the second generation switch designed by Pardee 2016.<sup>'''2'''</sup> | + | This part was designed using the tool designed by <span class="plainlinks">[https://2024.igem.wiki/wageningenur miRADAR]</span> for designing new toehold switches for miRNA detection, which is a modified version of the SwitchMi design model (UParis BME iGEM team 2021). The conserved regions and the structure of the toehold switch are based on the second generation switch designed by Pardee 2016.<sup>'''2'''</sup> |
For experimental data of this part, see <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106004 BBa_K5106004]</span>. This is a composite part, consisting of toehold switch A, under a T7 promoter and terminator, regulating the LacZ reporter gene. This composite part is tested in a cell-free (PURExpress) system. | For experimental data of this part, see <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5106004 BBa_K5106004]</span>. This is a composite part, consisting of toehold switch A, under a T7 promoter and terminator, regulating the LacZ reporter gene. This composite part is tested in a cell-free (PURExpress) system. | ||
==NUPACK Structure Analysis== | ==NUPACK Structure Analysis== | ||
| + | To check whether the designed toehold switch sequence does indeed form the expected hairpin structure, which gets linearized after hybridisation to the miRNA trigger, we analysed the structure using NUPACK software. NUPACK is a web-based software which predicts how two sequences hybridize based on base-pairing and mininum free energy (MFE) calculations.<sup>'''3'''</sup> NUPACK generates a figure which shouws the predicted secondary structure of the hybridised miRNA and toehold switch | ||
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| − | <small>'''Figure 2:''' Structure analysis at 37 °C using NUPACK cloud alpha software | + | <small>'''Figure 2:''' Structure analysis at 37 °C using NUPACK cloud alpha software,<sup>'''3'''</sup> with the different domains of the toehold switch annotated in the figure. a) MFE structure of toehold switch A without trigger RNA. b) MFE structure of toehold switch A hybridized to hsa-miR-484 trigger miRNA.</small> |
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| + | It can be seen that the toehold switch forms the expected hairpin structure, where the RBS is present at the top of the hairpin structure, and the start codon forms a bulge on the stem (Figure 2a). In addition, the miRNA trigger can completely hybridise to the toehold switch, resulting in partial linearization of the hairpin structure (Figure 2b). | ||
Latest revision as of 14:02, 29 September 2024
Toehold switch A for detection of hsa-miR-484.
This toehold switch is designed for detection of hsa-miR-484 (BBa_K5106000), a miRNA which is shown to be present at increased levels in the presence of multiple sclerosis. This is the first of two toehold switches, designed with an improved version of the SwitchMi design model (UParis BME iGEM team 2021), modified by miRADAR (WageningenUR 2024).
Introduction
A toehold switch is an RNA structure present on the 5’-UTR of a messenger RNA that prevents translation of a gene until a specific trigger RNA is present. This is due to the secondary structure of the switch RNA, which consists of a hairpin structure. This hairpin is formed by the ribosome binding site (RBS) and start codon (AUG), forming a loop and bulge respectively (Figure 1). After this hairpin, a 21-nucleotide linker sequence and the mRNA of an output gene are present. The toehold domain of the switch RNA is complementary to the trigger RNA, allowing for linear RNA-RNA interactions between the two.1
Figure 1: a) General structure of a toehold switch RNA, consisting of a complementary toehold domain, a hairpin structure formed by the ribosome binding site and start codon, and a repressed gene. b) Simplified mechanism of a toehold switch. The trigger (mi)RNA, which is complementary to the toehold region, binds to the switch RNA (depicted in yellow), allowing the hairpin to linearize. This allows for binding of the ribosome and translation of the repressed mRNA
When the trigger RNA is not present, the RBS is in the hairpin structure, disabling the possibility for the ribosome to bind, and thereby preventing translation of the protein. However, when the trigger RNA is present, it can bind the toehold domain of the switch, thereby opening the hairpin structure by a strand displacement reaction. This exposes the RBS and start codon, allowing ribosome binding and subsequent mRNA translation (Figure 1b).
Usage & Biology
This part codes for a toehold switch that contains a region that is complementary to the microRNA hsa-miR-484 (BBa_K5106000), which is upregulated in the presence of multiple sclerosis. In the presence of this miRNA, this trigger can bind to the toehold switch RNA, allowing for translation of a downstream protein coding region. This part can regulate expression of any protein in response to the presence of hsa-miR-484.
This part was designed using the tool designed by miRADAR for designing new toehold switches for miRNA detection, which is a modified version of the SwitchMi design model (UParis BME iGEM team 2021). The conserved regions and the structure of the toehold switch are based on the second generation switch designed by Pardee 2016.2
For experimental data of this part, see BBa_K5106004. This is a composite part, consisting of toehold switch A, under a T7 promoter and terminator, regulating the LacZ reporter gene. This composite part is tested in a cell-free (PURExpress) system.
NUPACK Structure Analysis
To check whether the designed toehold switch sequence does indeed form the expected hairpin structure, which gets linearized after hybridisation to the miRNA trigger, we analysed the structure using NUPACK software. NUPACK is a web-based software which predicts how two sequences hybridize based on base-pairing and mininum free energy (MFE) calculations.3 NUPACK generates a figure which shouws the predicted secondary structure of the hybridised miRNA and toehold switch
Figure 2: Structure analysis at 37 °C using NUPACK cloud alpha software,3 with the different domains of the toehold switch annotated in the figure. a) MFE structure of toehold switch A without trigger RNA. b) MFE structure of toehold switch A hybridized to hsa-miR-484 trigger miRNA.
It can be seen that the toehold switch forms the expected hairpin structure, where the RBS is present at the top of the hairpin structure, and the start codon forms a bulge on the stem (Figure 2a). In addition, the miRNA trigger can completely hybridise to the toehold switch, resulting in partial linearization of the hairpin structure (Figure 2b).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- Green, Alexander A., Pamela A. Silver, James J. Collins, and Peng Yin. ‘Toehold Switches: De-Novo-Designed Regulators of Gene Expression’. Cell 159, no. 4 (November 2014): 925–39. https://doi.org/10.1016/j.cell.2014.10.002.
- Pardee, Keith, Alexander A. Green, Melissa K. Takahashi, Dana Braff, Guillaume Lambert, Jeong Wook Lee, Tom Ferrante, et al. ‘Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components’. Cell 165, no. 5 (19 May 2016): 1255–66. https://doi.org/10.1016/j.cell.2016.04.059.
- Fornace, Mark E., Jining Huang, Cody T. Newman, Nicholas J. Porubsky, Marshall B. Pierce, and Niles A. Pierce. ‘NUPACK: Analysis and Design of Nucleic Acid Structures, Devices, and Systems’. Preprint. Chemistry, 11 November 2022. https://doi.org/10.26434/chemrxiv-2022-xv98l. https://www.nupack.org