User:ElNando888/Switch Cloud Labs/Chirality

Introduction

The first round of the Switch Cloud Labs was rich of surprises. I would put among the most interesting experimental results, the general failure in Lines by Satrryjess and the intrguing SHAPE data obtained in Chirality by kcabral28. I'd like to develop on the latter, and while I'll focus on the top-runner design, Brourd's Chirality - Sub 3 (unstable), I believe that the ideas presented below may very well apply to the vast majority of designs submitted in that lab.

The unbound state

At first sight, nothing looks strange in this design, but on a closer look on the terminal stacks, one can notice a few details.


Not overly stable	Simply unstable	Potentially stable

From these pictures, one may conclude that for both stacks to form, they basically had to do so simultaneously. Which would not be so unlikely, if the loop was symmetric, but with this difficulty added, the possibility seems quite remote to me...

Which is why I tend to believe in another hypothesis, which has the additional advantage to explain some of the experimental data directly. Check this out:

This is also potentially stable, and there are no problems with simultaneity, since it's a single stack. Placed in context, this gives:

The advantage I was talking about, is simply that this structure is fully consistent with the absence of SHAPE signal between bases 46 and 55.

You are probably going to object that this doesn't explain the signals around 22-24 and 29-34. Well, indeed, I don't have a solid explanation for those, but I do have a conjecture, supported by a simulation cooked by RNAComposer. Of course, RNAComposer's simulations don't have the same value as solved structures, like those found in PDB, but one could say that its depiction of stacks is probably very close from reality, while its generated loops (those that don't match precisely a fragment found in PDB) are well-educated guesses.

This said, please observe that a hook is actually a wine bottle opener, and that a hairpin loop has nothing to do with a balloon.

The stackish blob that turns magenta, yes, that's the hairpin loop.

The 45-56 bulge seems to be causing a 180° U-turn.

The second spin with volume added is meant to illustrate that:

there may be tertiary interactions between bases in the loop and the neighboring stack
and even without interactions, the proximity of the structures may make it very difficult for the SHAPE reagent to reach some of the binding points on the riboses, making those bases appear as protected, while they aren't actually paired.

The bound state

Apparently, your regular aptamer. But here too, the terminal stacks are actually a little weak.


Not stable	Smart, but not enough

Obviously, Brourd knew (or had a brilliant intuition) that those stacks would need some "help". It seems though that it was not enough, or that things didn't go as planned, since bases 50-52, strangely anough, show up as reactive...

At this point we can ask ourselves, how does the FMN complex form? From another type of ligand, theophylline, and this scientifix paper, it seemed natural to assume that both the upstream and the downstream stack would have to form first, before the docking can occur. If the downstream stacks are unstable and unlikely to form, how can it work?

-- Quizz mode on --

Quizz show host: « What does the FMN complex look like? »

Dumb candidate: (smashes button) «Like a bulging balloon »

(* BUUZZZZZ *)

Public: awww...

Quizz show host: « I'm sorry, wrong answer. It's a stack »

-- Quizz mode off --

So, now let's consider the hypothetical situation where the upstream stacks are formed, and the downstream ones are not. RNAComposer arrives at following conclusion (usual disclaimers)

To me, this suggests that the binding site (bases in magenta) could actually easily form spontaneously, should a FMN molecule pass by.

Unfortunately, in the case of the bound state, I don't have a clear picture of what may have happened to the downstream bases. I suspect though, that they didn't fold as planned, and that, just like in the unbound state, some form of tertiary interaction happened. The strongest clue in favor of this hypothesis is the SHAPE data for base 21.

The base 21 typically shows as reactive. That's the normal case for a binding site in complex with FMN. Here though, it shows as protected...

Afterthoughts

(coming soon)