You developed a computational pipeline to design 'toehold' riboswitches for specific RNA trigger sequences. The pipeline predicts which toehold switch design adheres to the secondary structure of a known toehold switch, through a combination of RNA secondary structure prediction and subsequent free energy prediction of that structure to assess its stability. A further in silico validation of the method via molecular dynamics is included.

While the method is in itself well designed and likely very helpful in relation to toehold switch design, the interpretation of the in silico results is overly optimistic. Typically, in silico methods are very good at separating what might work from what likely does not work (in this case, a particular toehold switch), but here there is no assessment of how reliable your method is, as validated by experimental data. This is an essential component that is missing in your study: is there (independent) data available on toehold switches that are know to work, and is your approach able to detect/score those? Do you have any independent experimental data that illustrates that this method in fact works well (you mention the iGEM project A.D.N. - did this use your approach, and if so how well did it work)?

In addition, RNA is a notoriously flexible molecule issue, how well can the silico RNA predictions that you are using account for that  - do your ‘free energy’ calculations take entropy into account? What could go wrong in these calculations? These issues are not addressed - but should be.

Finally, your statements should always mention ‘predicted’ when this is where your information comes from, e.g. page 10, section 3.2, should say ‘predicted secondary structure’ - as this is what it is, there is no experimental validation.

You should address these issues, or tone down your statements about the real-life applicability of your method, as at the moment it is impossible to assess whether your pipeline works in reality (or not).

I found the preprint clear well written, with only a few typos and formatting issues (listed below). As a non-specialist reviewer, there were two terms that I found difficult to understand and I recommend that the authors include a few words of explanation about each of them in the paper to make it more accessible to a broad readership: "orthogonality" and "overregard". I also found some sentences that sounded quite finalist, and recommend reformulating them to avoid this: "nature has explored many different regulatory mechanisms"; "capable of regulating transcription and translation to optimize the use of resources" (I suggest "capable of regulating transcription and translation, thereby optimizing the use of resources" that does not convey this finalist undertone).

Minor typos and formatting issues:

1) please put line numbers in your resubmitted preprint - it is tricky to provide feedback without them

2) in part 1.1., "RNA molecules, which typically" should be "RNA molecules that typically"

3) "have led to several different designs.-" please remove the unnecessary hyphen

4) "a ribosome binding site" -> "a ribosome-binding site"

5) in panel 3.2 of Figure 2, "cannonical" -> "canonical"

6) in part 3.1, "did not unwind which would lead" -> "did not unwind, which would have lead"

7) in part 4.1, "was stable in the conditions" -> "was stable under the conditions"

8) "with their complementary sequences fluctuates the most often during the simulation" -> "with their complementary sequences fluctuated most often during the simulation"

9) "presented a similar structure to the one" -> "presented a structure similar to the one"

10) "All switches have a negative energy that predicts" -> "All switches had a negative energy that predicted"

11) nearly all references are in "sentence case" except three that are in "Title Case" (refs. 1, 9 and 22), please put them in "sentence case" as well

12) please remove "Chapter one - " from the begining of the title of reference 22, and please remove also the number "1" at the end of the title

13) in the PDF preprint, clicking on the references link to a paperpile URL that gives an error message. Please remove these useless links or replace them with proper DOI links.