Player Experiment Challenge 1

Within the following Google document is an experimental challenge for players, to design experiments to test the thermodynamic stability of A-U rich helices in an RNA ensemble. Using the Das Lab cloud lab platform.…

This is an experiment to ascertain whether or not those players who want to participate in the lab are willing to do so, given the goal and opportunity.

We can choose which targets and experiment protocols are the best for achieving the goals in this Google document, by voting here in Getsatisfaction.


Here is a picture of what would be given to players in the lab. This is a solution to the “pincers” switch from the challenges where the 2nd state has 0CG.…

My hypothesis is that the state with the more CG will be the one that is predominant in the ensemble - State 1 with 2 CG in it.

The 2nd state is not stable without a molecule but it is my understanding that it will appear some of the time even without a molecule.

Players will be limited in the number of mutations (say 10-20) that they can make to this lab such that an AU pair could be made into a CG pair with 2 mutations.

For example:…

with 4 mutations a player could design this sequence giving the second state 2CG and the first state 3CG. the ratio of the CG count in the states is now 3:2 instead of 2:0.

While state 1 is still stable and state 2 is not, my hypothesis is that state 2 will be more prevalent than it was before. Should state 2 be given more CG than state 1 my hypothesis would be that it would become more prevalent in the ensemble.

(This example ignores the collapsing of the aptamer that players can mutate away if they wish and of course any switch puzzle/lab could be substituted for “pincers” with the same idea.)

Not sure if this fits the experiment set by Brourd but i have been looking at Brourd’s TEP Riboswitch Targets Lab and have noticed that for the vast majority of the designs there is a strong affinity for the two way junction to form over the three way. An example of the SHAPE suggesting the pairing to the other six nucleotides in state 2 ( ). This affinity for the two way junction is even evident in the sublab Shift 7x5 Riboswitch where in the three way junction - State 1- there is a 7bp stack switching to a two way junction with a 5bp stack in State 2.

It is intriguing to me that a three way junction, even with a 7bp stack and a tetraloop as the hairpin loop is difficult to form even 50% of the time based on the results of the SHAPE reactivities. i am curious what would happen if we limit the number of GC’s to perhaps just one in both states (or perhaps even all AU helices) and incrementally increase the basepairs in the hairpin for the three way junction against the two way in state 2. I.E. make a Shift 8x5, 9x5 or 7x4 Riboswitch. I would continue to use the TEP Aptamer sequence in State 2 used in Brourd’s labs as a baseline for comparison.

In theory, there would be multiple sequences to test out. such as all AU helices, or GU’s incorporated into the switches or allowing a single GC pair in either or both states. What would it take to swing the SHAPE reactivities so that the three way junction is formed approximately 50% of the time? Can it be done with an all AU helix?

I wonder if an experimental series coud be developed tat tests the CG ratio between the states, enforcing ratios, starting at, say 5:0, 4:0…0:0, then 5:1, 4:1, …0:1, up to 5:5, 4:5, …0:5, unless we are not interested in ane experiments with more CG in state 2, in which case this series would lose the second lower right “quadrant” (any state1 CG< state2 CG).