This repository contains the code for Toehold Design based on the NUPACK package[1]. The program was built as part of the BioPank project for the iGEM 2021 Contest. The iGEM UNILA_LatAm implemented this code to design adapted Toeholds for *Bacillus subtilis* in the detection of miRNAs acquired from the extracellular environment in the *Lutzmyia longipalpis* midgut (miR-283).
The project was uploaded on GitHub because of iGEM’s rule
Carpincho Tool segue estritamente o modelo Type II descrito por Green et al.(2020) [2] e baseia seu funcinamento em NUPACK Design and Analysis algorithms.
- NUPACK 4.0.0.27 (http://nupack.org/downloads)
- Biopython v1.79
- NumPy v1.21.1
- Pandas v1.3.1
- Matplotlib v3.4.2
- Forgi v2.0.2
- RNA v0.7.5
- In the root directory:
mk dir build
cd build
cmake ../
install the libraries versions (pip3 install (library name)... == version )
- Dowload Carpincho Toehold Designer (Git link) and go to the root directory and type
python3 Carpincho.py
You can fill the inputs for your Toehold Design preferences in th Input.py file. The variables are:
| Variable | Description |
|---|---|
| Unpaired | Seed sequence for trigger binding that is not connected to hairpin |
| Paired | Lower stem nucleotides |
| Prevent | Sequence patterns to prevent |
| StopCodons | Stop codons to Prevent between RBS and Linker |
| Loop | RBS sequence (with or without spacers) |
| Trials | Number of iterations for Toehold generation |
| Trials_stop | Number of iterations if stop codons are identified |
| Material | Temperature-dependent RNA and DNA free energy parameter sets (NUPACK User Guide |
| T | Temperature specified (default: celsius = 37) (NUPACK User Guide |
| Sodium | Salt Conditions default = 1M (NUPACK User Guide |
| Magnesium | Salt Conditions default = 1M (NUPACK User Guide |
| Wobble | True- do not follow WATSON-CRICK base pair rules (G-U, for instance) |
| miRNA (trigger) | Sequence Trigger for miRNA target |
| Linker | Linker sequence |
| Reporter | Reporter (firstest 5 codons) |
| PATH | Directory output path |
After installation and running, Carpincho Tool saves each version in a folder that contains subversions if there are stop-codons (Trials_stop). These sub-versions are stored in version.sub-version folders.
- Binding_Toehold_Trigger.fx: Trigger+Toehold sequence and Structure notation;
- Toehold_version.fx: Toehold sequence and Structure notation;
- Toehold_version_struct.png: Toehold conformation from ViennaRNA package[3];
- Binding_Toehold_Trigger_probab.png: Pair binding probabilities
- Binding_Toehold_Trigger_probab_Theory.png: Pair bindgin probabilities for toehold+trigger binding structure in theory (standard)
- Toehold_version_probab.png: Pair probabilities for Toehold Structure
- Toehold_version_probab_Theory.png: Pair probabilities for Toehold Structure in theory
- .csv file: All thermodynamic parameters, ensemble defects, sequences and structures information
ResultsToehold_Bs_RBS.zip shows an example of output files. This Toehold were designed for B. subtilis to detect miR-283
- Limited by the standardized structure at unpaired =11 and paired = 10
- Slow for large libraries
- The code does not contain the best toeholds score
[1] J. N. Zadeh, C. D. Steenberg, J. S. Bois, B. R. Wolfe, M. B. Pierce, A. R. Khan, R. M. Dirks, N. A. Pierce. NUPACK: analysis and design of nucleic acid systems. J Comput Chem, 32:170–173, 2011.
[2] Green, A.A. et al. (2020). Complex cellular logic computation using ribocomputing devices. Nature, 548, p. 117-121. [3] Lorenz, Ronny and Bernhart, Stephan H. and Höner zu Siederdissen, Christian and Tafer, Hakim and Flamm, Christoph and Stadler, Peter F. and Hofacker, Ivo L. ViennaRNA Package 2.0. Algorithms for Molecular Biology, 6:1 26, 2011, doi:10.1186/1748-7188-6-26