Part:BBa_K5106001
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.
Figure 1: a) General structure of a toehold switch RNA, containing 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.
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
Figure 1: Structure analysis at 37 °C using NUPACK cloud alpha software. a) MFE structure of toehold switch A without trigger RNA. b) MFE structure of toehold switch A hybridized to hsa-miR-484 trigger miRNA.
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]
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