Lab protocols and folding kinetics

I tried to find the answer to my question in the documents listed on the Das Lab page, but couldn’t find it. Essentially, I’m interested in the exact environmental conditions applied to the RNA strands right before the introduction of the chemical probes.

What I think I know already, is that the RNA is first synthesized from the DNA templates, with a well-known procedure called PCR. Then, I suppose that the purified products are stored.

Then comes the chemical modification. If I understand correctly, the RNA is first denatured by heating it to 90°C. Then… well, that’s the missing link for me. When I look at RMDB entries, I see annotations about 24°C… Ok, that’s room temperature. So, does the experimenter let the solution cool down at room temperature, for say 4 hours, at which point, there is no doubt that the RNA has completely folded, and adds the SHAPE reagent at that moment? Or does the experimenter monitor the cooling solution, and does s/he add the chemical probe right the instant the solution passes the 37°C point? If so, how long does it take? Or is there any form of “speeded up” cooling at any point? In short, what is the evolution of the temperature of the solution over time, until the point where the chemical probe is introduced?

I’m asking because I intend to revisit the work that was done 13 years ago and presented in this scientific paper. The software is still available as part of the Vienna package, and includes computing folding trajectories for growing chains (essentially attempting to reproduce transcription) and the so-called “snap-cooling” (the chain is assumed to be complete in length, and unfolded) But I noticed that the simulation options do not include a varying temperature. I think I understand that this was left aside at the time (in 2000) for performance reasons. Maybe it could be possible to find powerful enough hardwares today, which could sustain the heavy computational load.

Side question: if it’s actually 24°C, why do we use 37°C in our in silico simulations on EteRNA?

90°C for 1 minutes in a buffer with pH 6.0. [to help refold secondary structure]

then add MgCl2 up to 10 mM at room temperature, and
then 50°C for 30 minutes [conditions that help even complex RNAs (such as the group I self-splicing intron) find their functional structures]

cool to room temperature on bench top, and add a buffer (50 mM Na-HEPES) that raises pH to 8.0 as is appropriate for SHAPE reaction.

SHAPE for 10 minutes at room temperature.

Comment – we didn’t used to do the 50°C incubation in general, but now we do after we noticed that a ‘control RNA’ (the P4-P6 domain of the Tetrahymena group I intron) was getting misfolded or staying partially unfolded in our cloud labs – as if the other 1000s of RNAs were glomming on. The 50 °C incubation did the trick.

Well, thank you very much, it clarifies many things.
Unfortunately for me, it also probably means that the goal of simulating folding trajectories as close to reality as possible, remains in the domain of the computationally rather impracticable… :confused: Maybe I’ll still give it a shot when I’ll be less busy, but I’m not holding my breath.

Yea. There are a lot of folks (including academic labs) who are interested in looking at time-resolved folding. One thing that my lab & collaborators are working on is a way to record ‘time’ in the sequence itself – if we get that method working we can share the data with you. Are there particular RNAs whose folding trajectories you would be most interested in?

Many. Any of our lab candidates which happen to clearly deviate from our “cherished” MFE. For those, I’d like to see if I can discover the reasons behind the apparent failure (probably a kinetic trap of some sort, and if not, then it would possibly be an indication of some “hole” or defect in the energetic model). And of course, all sorts of multistable sequences, conformational switches, etc. If I had the means of my “ambitions”, I’d like to work in the same direction as was outlined in this paper (which you probably already know), I was hoping to use a different method though.

And… woah! Record time in the sequence?? Mind… blown…
Well, I’d love to hear about it of course!

Here’s a hint – the method would be for watching RNA fold as it is actually being transcribed!

Sorry if I’m being dense here, but just to make sure:

The buffer is heated up from room temperature to 90°C and maintained there for at least 1 minute


The buffer is at room temperature and exposed to a bath at 90°C for 1 minute


The buffer (and the RNA which is in the buffer) is heated up from room temperature to 90°C and maintained there for at least 1 minute. To clarify further, I talked to Ann, and she has been using a 3 minute incubation.