Author Interview – Toeholder: a software for automated design and in silico validation of toehold riboswitches

PeerJ spoke to Angel F. Cisneros and Francois D. Rouleau about the recently published PeerJ Physical Chemistry article: Toeholder: a software for automated design and in silico validation of toehold riboswitches.

Francois Rouleau

Angel Cisneros

Can you tell us a bit about yourselves?

We are both graduate students at Université Laval. As part of a project for the 2019 edition of iGEM, we developed a tool to help design toehold riboswitches for any sequence of interest. After the competition, we felt that the tool could be of use to the scientific community at large, and we decided to improve it as much as possible and try to publish it in a peer reviewed journal.

Can you briefly explain the research you published in PeerJ?

We developed a tool to design toehold riboswitches for any sequence of interest. Briefly, toehold riboswitch is an RNA molecule that contains all the elements necessary for translation of a reporter gene (for example, green fluorescent protein). However, it folds into a particular secondary structure that blocks the access of the ribosome to its binding sequence, which usually blocks translation. The only way to allow translation is to unfold the secondary structure, which can be done in the presence of a DNA/RNA sequence that binds to a particular site (α & β, the trigger sequence of the toehold switch). As a result, toehold riboswitches can be used as sensors to detect particular sequences in a sample, since their presence leads to the translation of the reporter sequence.

Legend: Toehold switches are complex to manually design as they have many parts that must be precisely engineered. For instance, β’ depends on the sequence of β, and neither region can have complementarity to any other parts of the sequence.

Toehold riboswitches have many potential applications, but designing them is not always straightforward. When we first explored this concept, there were very few resources to help guide us in designing our switches. Thus, we aimed to develop a tool that would facilitate their design and eventual applications in the most effective and straightforward manner.

2D structures of typical toehold switches in OFF and ON states.

What did you discover and where?

We based ourselves on the experimentally published datasets that characterized toehold switches to try and go further in their analysis of what made a “good” toehold. Because of the limitations placed on us by our status as an iGEM team, with effectively no budget, we really focused on the modeling of the switches to try to find specific parts that were significatively different between “good” and “bad” switches. Using molecular dynamics, we identified parts of the switches that, based on the biophysical principles of how the toeholds work, could/should help design better switches.

What was significant about your findings?

What was the most significant part of this study was how we integrated the properties that we identified using our model within the Toeholder tool. By incorporating these positional properties into the Toeholder output ranking, we hope that we can help users make the best switches without having to go through extensive trial and error, as we did during the iGEM competition.

What kinds of lessons do you hope your readers take away from the research?

It’s not because a dataset has been published and a big paper was written using it that this dataset has been mined dry, to use a figure of speech. There is so much available data with untapped potential that the original authors moved on from. So much more information can be gleaned from this. When reading a paper, if you feel like you have a good idea or good insights that were not explored in the original paper, go dig in the data. Go try it yourself. You never know what you might stumble upon.

For iGEMers more specifically, we’d say to not undersell your own research. It’s not because the projects are student-led that the work is necessarily of lesser quality. If you are proud of your work and trust in the quality of your research, it is worth it to submit to peer review organisms and get some feedback, and maybe even get it published.

Do you have any comments about your overall experience with us?

Publishing with PeerJ by going through the PCI reviewing process went quite smoothly. The people at PeerJ rapidly answered all of our concerns, and as we were students with no PI on this project, even agreed to waive the APC fees to allow us to publish our research. We are very grateful for that, as we would not have been able to formally publish our research without their help.

Was this your first time submitting to the Peer Community In.. platform?

Yes, this was our first time submitting to the Peer Community In.. platform. We initially learned about it through a conference held in our research institute about transparent publishing/reviewing. We felt that this was perfect for what we had. Even if it would’ve stayed at the preprint step, we would at least have peer reviews to improve both the tool and the preprint, as well as to lend credibility to our work.

Would you recommend other researchers follow this process?

Yes, we believe Peer Community In… is doing a great job at aiming to democratize science. For example, researchers that are short of funding can submit their preprint to them to get a review. These reviews could help them have a higher probability of their manuscript being accepted once they submit it to their target journals. However, even if the paper was not accepted, the preprint would be accompanied by the review, which can be a good read for other researchers as they navigate through the original work. Thankfully, Peer Community In… is not alone, as other platforms like Review Commons and the journal eLife are moving in a positive direction towards democratizing science and making the peer review process more transparent.

Why did you choose to publish the article in PeerJ Physical Chemistry after you received the recommendation in PCI?

Looking through the list of PCI-friendly journals, PeerJ Physical Chemistry looked like the best fit for our study. We believed this journal was the best option for our manuscript since it had both the best fit for the topic and it would really look at all the hard work that we had already done with the previous PCI reviews.

How was the experience?

We are overall quite satisfied with the experience. We received very useful comments from the Peer Community In… platform that helped improve the quality of our research. With the changes and their review supporting us, we were able to submit to PeerJ Physical Chemistry and everything advanced smoothly. Publishing without a last author/principal investigator was a daunting task, and the people at PCI and PeerJ were an amazing help.

You can find more PeerJ author interviews here.