User talk:Brourd

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Revision as of 04:22, 21 February 2014 by Brourd (Talk | contribs)

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Just creating the page, use the '+' link above to start a new thread.



Hi Brourd,

I think it would be good to present the motivations behind the whole project. Since I'm not entirely sure I'm getting 100% of it myself, I'm going to write down what I understood, and I'd appreciate your correcting me when I'm wrong.


I believe both the scientists and the players would love to try designing switches again. There are issues though, related to the new pipeline, Cloud Lab.

  • it doesn't make sense to run switch-related sequences along with mono-state ones. It would represent a costly waste of resources
  • making a round with exclusively switching sequences is basically twice the work for the lab, and probably also twice the price


So, what can we do? Some time ago, an idea emerged, that could be used in conjonction with Cloud Lab as it is now, without disturbing the cycle too much. Essentially, the idea consists in submitting the same sequence twice, once with the FMN bindng site, once with that area mutated to something else, which we will dub as "mimic". Assuming the mutation would have the same effect as a real FMN binding with the aptamer (in other words, providing a bonus of about -4.9 kcal/mol), this system would allow us to screen for good switch candidates. And once we have accumulated enough of those, a dedicated lab run could be done, with the real FMN addition this time.

If I understand correctly our purpose in this project, our goals are:

  • first, to create good mimics
  • second, to start generating good switches


About the first step, I may have missed something, but I couldn't find any documents, neither on EteRNA, nor on RMDB, related to tests with mimics that Das Lab would have already made. The only thing I heard (from you Brourd) is that they tested this:

Mimic 1.png Mimic 2.png

Which I would argue, doesn't look very good to me, but let's leave this topic for later.


And once we have (or at least, we think we have) good enough mimics, the second step sounds almost "small and easy": design targets, and sequences for those. And voila :)

-- ElNando888 (talk) 15:07, 20 February 2014 (UTC)



So, the question of where the FMN mimic sequence originally started...

The FMN Mimic was actually run for 2 designs of every lab in Round 70 of the cloud lab, aka, the first and only round of FMN switches in the cloud lab so far.

As you may recall, I actually wrote something about it, as well as a question of the repeatability of the riboswitch scoring a very, very, long time ago.

In addition, Dr. Rhiju Das wrote a little blog post about how his Ph.D students participated in the first round of the EteRNA switches, and if I recall correctly, these switches were made and scored with the FMN mimic.

All the chemical mapping data for the FMN switches and mimics are available in rounds 70 and 71 on the RMDB. I'm sure you can figure out what to do from here!


So, back to the motivation and goals for the project, and why they were not included in the original post.

It was 1 am :P (I'll work on including that)

However, your observation is correct. The goal of the FMN mimic is to essentially create a protocol, where a multiple state RNA system based on the binding of a ligand, can be potentially tested in absence of said ligand (other multistate systems may be a tad more difficult). This protocol could potentially extend to other other aptamers as well, which would be a future goal. in addition to this, this project has two additional main goals, as well as a few secondary goals

  1. The characterization and creation of successful riboswitch constructs.
  2. The characterization and implementation of successful riboswitch design rules.
    1. Potential reworking of riboswitch scoring based on the SHAPE chemical mapping protocol.
    2. A comparison of riboswitches with canonical base pairs versus those with noncanonical base pairs.
    3. Implement a pipeline for the testing of riboswitches using the Das Lab's high throughput chemical mapping protocol.
    4. Determine if current automated algorithms can design successful riboswitches (NUPACK, ViennaUCT, any other publicly available multistate design algorithms) 
      1. (A VERY minor/secondary goal) The creation of an automated algorithm to design riboswitches, coding both the rules for constructs and sequences into it.


-- Brourd (talk) 22:51, 20 February 2014 (UTC)



Thanks Brourd,

The data I can find in round 70 on RMDB indicates that only and all EteRNA players' designs were tested 4 times, with and without FMN, with 2 different chemical probes, 1M7 (SHAPE) and DMS. I see no traces of mimics in the dataset.

Round 71 (Cloud Lab round 3) was a repeat of Cloud Lab 1, so completely different sequences and constructs. Though, this batch does include the students switch constructs Rhiju is talking about. But those sequences weren't tested against the real FMN...

So, I still don't see any data that could speak about the effectiveness of the method for screening good FMN switches.

-- ElNando888 (talk) 03:32, 21 February 2014 (UTC)



So, in round 70, annotation data 3713 to annotation data 3872. The map-seq ID has the ID, then -a, to indicate that it is a mimic. (a for alternate, maybe?)


ANNOTATION_DATA:3868   modifier:DMS MAPseq:design_name:JG #1 MAPseq:project_name:Top Notch by jmf028 MAPseq:ID:2426211-a  



signal_to_noise:weak:0.325 MAPseq:tag:FAM-RTB003 chemical:FMN:200uM


The FMN mimic is in bold in the sequence.


---Brourd (talk) 04:22, 21 February 2014 (UTC)

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