A novel approach for engineering biological systems by interfacing computer science with synthetic biology
Toeholder: a Software for Automated Design and In Silico Validation of Toehold Riboswitches
Recommendation: posted 15 December 2022, validated 16 December 2022
Melamed, S. (2022) A novel approach for engineering biological systems by interfacing computer science with synthetic biology. Peer Community in Genomics, 100157. https://doi.org/10.24072/pci.genomics.100157
Biological systems depend on finely tuned interactions of their components. Thus, regulating these components is critical for the system's functionality. In prokaryotic cells, riboswitches are regulatory elements controlling transcription or translation. Riboswitches are RNA molecules that are usually located in the 5′-untranslated region of protein-coding genes. They generate secondary structures leading to the regulation of the expression of the downstream protein-coding gene (Kavita and Breaker, 2022). Riboswitches are very versatile and can bind a wide range of small molecules; in many cases, these are metabolic byproducts from the gene’s enzymatic or signaling pathway. Their versatility and abundance in many species make them attractive for synthetic biological circuits. One class that has been drawing the attention of synthetic biologists is toehold switches (Ekdahl et al., 2022; Green et al., 2014). These are single-stranded RNA molecules harboring the necessary elements for translation initiation of the downstream gene: a ribosome-binding site and a start codon. Conformation change of toehold switches is triggered by an RNA molecule, which enables translation.
To exploit the most out of toehold switches, automation of their design would be highly advantageous. Cisneros and colleagues (Cisneros et al., 2022) developed a tool, “Toeholder”, that automates the design of toehold switches and performs in silico tests to select switch candidates for a target gene. Toeholder is an open-source tool that provides a comprehensive and automated workflow for the design of toehold switches. While web tools have been developed for designing toehold switches (To et al., 2018), Toeholder represents an intriguing approach to engineering biological systems by coupling synthetic biology with computational biology. Using molecular dynamics simulations, it identified the positions in the toehold switch where hydrogen bonds fluctuate the most. Identifying these regions holds great potential for modifications when refining the design of the riboswitches. To be effective, toehold switches should provide a strong ON signal and a weak OFF signal in the presence or the absence of a target, respectively. Toeholder nicely ranks the candidate toehold switches based on experimental evidence that correlates with toehold performance (based on good ON/OFF ratios).
Riboswitches are highly appealing for a broad range of applications, including pharmaceutical and medical purposes (Blount and Breaker, 2006; Giarimoglou et al., 2022; Tickner and Farzan, 2021), thanks to their adaptability and inexpensiveness. The Toeholder tool developed by Cisneros and colleagues is expected to promote the implementation of toehold switches into these various applications.
Blount KF, Breaker RR (2006) Riboswitches as antibacterial drug targets. Nature Biotechnology, 24, 1558–1564. https://doi.org/10.1038/nbt1268
Cisneros AF, Rouleau FD, Bautista C, Lemieux P, Dumont-Leblond N, ULaval 2019 T iGEM (2022) Toeholder: a Software for Automated Design and In Silico Validation of Toehold Riboswitches. bioRxiv, 2021.11.09.467922, ver. 3 peer-reviewed and recommended by Peer Community in Genomics. https://doi.org/10.1101/2021.11.09.467922
Ekdahl AM, Rojano-Nisimura AM, Contreras LM (2022) Engineering Toehold-Mediated Switches for Native RNA Detection and Regulation in Bacteria. Journal of Molecular Biology, 434, 167689. https://doi.org/10.1016/j.jmb.2022.167689
Giarimoglou N, Kouvela A, Maniatis A, Papakyriakou A, Zhang J, Stamatopoulou V, Stathopoulos C (2022) A Riboswitch-Driven Era of New Antibacterials. Antibiotics, 11, 1243. https://doi.org/10.3390/antibiotics11091243
Green AA, Silver PA, Collins JJ, Yin P (2014) Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell, 159, 925–939. https://doi.org/10.1016/j.cell.2014.10.002
Kavita K, Breaker RR (2022) Discovering riboswitches: the past and the future. Trends in Biochemical Sciences. https://doi.org/10.1016/j.tibs.2022.08.009
Tickner ZJ, Farzan M (2021) Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors. Pharmaceuticals, 14, 554. https://doi.org/10.3390/ph14060554
To AC-Y, Chu DH-T, Wang AR, Li FC-Y, Chiu AW-O, Gao DY, Choi CHJ, Kong S-K, Chan T-F, Chan K-M, Yip KY (2018) A comprehensive web tool for toehold switch design. Bioinformatics, 34, 2862–2864. https://doi.org/10.1093/bioinformatics/bty216
The recommender in charge of the evaluation of the article and the reviewers declared that they have no conflict of interest (as defined in the code of conduct of PCI) with the authors or with the content of the article. The authors declared that they comply with the PCI rule of having no financial conflicts of interest in relation to the content of the article.
Evaluation round #2
DOI or URL of the preprint: https://doi.org/10.1101/2021.11.09.467922
Version of the preprint: 2
Author's Reply, 09 Dec 2022
Decision by Sahar Melamed, posted 02 Dec 2022, validated 05 Dec 2022
Thank you for resubmitting your manuscript entitled "Toeholder: a Software for Automated Design and In Silico Validation of Toehold Riboswitches". You have successfully addressed all the comments from the reviewers. However, before we recommend on this manuscript there are minor revisions that are needed.
- Figure 2: The text is smaller in comparison to the other figures. Please enlarge it.
- Please consider dividing the "4.2 Toeholder conception and validation" subtitle into two subtitles.
- 7.Data availability: please add the DOIs of your data, scripts and code in this section.
- Please add a section "10. Supplementary material" with the DOI/URL to your supplementary files (Supplementary video 1).
Evaluation round #1
DOI or URL of the preprint: https://doi.org/10.1101/2021.11.09.467922
Version of the preprint: 1
Author's Reply, 07 Nov 2022
Decision by Sahar Melamed, posted 13 Jul 2022
Dear Dr. Cisneros,
Thank you for the opportunity to consider your manuscript at PCI Genomics. The two reviewers found the work interesting but also raised some concerns. We would like to invite you to submit a revision if you can address the reviewers' key concerns, especially those raised by reviewer 1. The main comment of reviewer 1 is related to the interpretation of the in silico results. Please consider if you can add experimental data to support the method or alternatively revise the text so it will better represent the applicability of the method.
We hope that the comments below will prove constructive, and I would be happy to discuss any plans for a revision once you've had a chance to consider the points raised by the reviewers.
Sahar Melamed, PhD
Recommender at PCI Genomics