User talk:Brourd

From EteRNA WiKi
Revision as of 22:51, 20 February 2014 by Brourd (Talk | contribs)

Jump to: navigation, search

 

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

 

Background

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)

Personal tools
Main page
Introduction to the Game