Part:BBa_K3905004

gen2 517-5p Toehold Switch

This Toehold Switch is activated by hsa-MiRNA 517-5p. Toehold switches contain multiple parts, namely: the trigger binding site, the ribosome binding site (RBS), the seven amino acid long linker sequence and the protein coding sequence.

The trigger binding site on each toehold switch is the reverse complement to the trigger RNA strand. As the miRNAs are both 22 nucleotides long, the trigger site is also 22 nucleotides long. The trigger binding site makes up the lower stem in the ‘off’ state as well as part of the toehold base. We were advised to ensure the trigger site was >10 nucleotides long by Dr Wooli.

When few microRNAs are present, the ribosome binding site of an mRNA coding for luciferase is folded up, so the ribosome cannot unzip the long stem and move down the strand to the start codon to initiate translation.

However, when a microRNA binds to the trigger site, the switch unfolds, decreasing the length of the stem, and exposing the start codon, so translation of luciferase - a reporter protein - can begin.

This was designed using a python script taken from the NUPACK API, there is more information about this here https://2021.igem.org/Team:City_of_London_UK/Software .

Labelled toehold switch in its 'off' state.

Characterisation

methodology

For each gen2 switch, we tested, at a constant DNA concentration, the target miRNA (miR-517-5p or miR-210-3p) at 9M and 2.25M to replicate the roughly 4-fold increase of expression levels in patients with preeclampsia 1 2, as well as a homolog for each miRNA (miR-518f-5p for miR-517-5p and miR-6867-5p for miR-210-3p), which were determined to be the miRNAs have the highest probability of activating the switches aside from the target miRNAs themselves by our software tool (see software page). We then incubated the DNA, miRNAs with a plasmid containing a gene coding for T7 polymerase (to promote transcription of our toehold switch mRNAs) as well as a TXTL cell-free master mix for 1 hour, and then pipetted the contents of each tube into 8 wells of a 96 well plate, and took four measurements after D-luciferin in a buffer was injected, at different times.

results

Graph comparing the luminescence against the concentration and specie of miRNA.

Graph comparing the luminescence against the concentration and specie of miRNA in respect to the negative control.

Graph comparing the luminescence against the concentration and specie of miRNA over time.

The results show a brief, high peak in luminescence for a high concentration of miR-517-5p. This is because, during the incubation time, more luciferase is produced due to an increased rate of translation, so there is a higher concentration of luciferase, so more luciferase-luciferin complexes form at the beginning, converting luciferin to oxyluciferin, producing luminescence. The results show that, compared to the negative control, the homolog ‘miR-518f-5p’ activates the switch more, causing an higher peak, sooner on, and there is only a small increase in luminescence between the control and the target RNA at reduced concentration. Based on this data, we decided that our kit should detect luminescence at some point between 100 and 200 seconds. These diagrams show how the toehold switch can discriminate clearly between miR-517-5p at 9M and 2.25M.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 15
  Illegal XbaI site found at 53
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 15
  Illegal XbaI site found at 53
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 15
  Illegal XbaI site found at 53
  
• 1000
  COMPATIBLE WITH RFC[1000]