What is Free Energy?

When you are playing the puzzles in EteRNA, there is a box in the top left corner which displays the [free] energy of the current molecule. What that is a measure of is the potential energy of the molecule. Everything (at temperatures above 0 K) has some amount of internal energy. When you look at energy, LOWER ENERGY IS (generally) BETTER, meaning larger negative numbers.

In the world of protein folding and RNA folding, the general theory is that the lowest energy state is the most stable. However, this does not necessarily mean that when designing a solution for the RNA lab, you should try to get the lowest possible free energy. There are other things to consider as well, which are discussed in the RNA Lab Guide.

A note on units:
The energy is given in units of kcal which stands for kilocalories. For more on this, read the Wikipedia article on kcals kcals and kcals/mol.

Further Reading:


How does the program calculate free energy?

Internally, EteRNA uses the ViennaRNA energy function. Some players have been reverse engineering parts of the energy function. You might find this post about stacks interesting, and this one about loops.

What is the significance of the Yellow score that appears after each puzzle completion?
Also what is the significance of the little box on the left that says something like:
-199.1 kCal

This reply was created from a merged topic originally titled
Yellow score and kCal.

Thanks tor the reply to the 2nd part of my question.
The 1st part was what is the significance of the yellow score
that appears after the white score or rank.

More tutorials needed please. No information is given about the calories reading. I add the needed pairs, but the total pairs count goes down. It seems to matter where I place the pairs, but this is not explained anywhere.

An explanation of what I am trying to do is needed. I know I need to make the stacks white and have the required number of a certain pair, but sometimes changing to the required pair makes the stack turn red. I randomly change bases until it works, but I don’t know why it works. What re the rules? What is required to make a stack white?

This reply was created from a merged topic originally titled
More tutorials needed please..

Cool – thanks.

able to see the free energy of the synthesized lab design, so we can compare what and why it behaves like that.

This reply was created from a merged topic originally titled
Free energy should be displayed on the lab results.

Is there any guidance on melting point and its interaction with free energy? I note dimension9’s comments on Improved Statistics Display in Lab Graphics (wherein these
were discussed as added tools):

From Recent RNA Labs
Temp Melt -> Free Energy Range
107C -30 -> -87.8 kC
97C -27.9 -> -46.6 kC
77C -13 -> -28.6 kC
67C -9.8 -> -28.2
57C -10.4 -> -12.5

Anyway, still confused and wondering how to usefully employ melting point in addition
to free energy- much appreciated

This reply was created from a merged topic originally titled
Melting Point and Free Energy.

I agree! Granted, I’m brand new to EteRNA, but I played through the tutorial and don’t recall hearing anything about how energy should factor into the strategy.

It would be great if there were a tutorial about nucleotide numbers and free energy, how they impact total energy, and most importantly, their impact on how the RNA folds. I assume energy has something to do with how some pairs bond or unbond when I make a change to another part of the molecule, but I have no idea how it works. …It’s a big de-motivator.

I wonder about that also. What intrigues me is seeing how changing nucleotides in a distant arm of a complex molecule can stabilize or destabilize a remote area in the whole structure. Free energy has got to play a role in this or the levels of energy in various loops and stacks but I have not yet found workable patterns. I am still solving mostly by trial and error.

Free energy can play a role in stabilization, particularly when the overall free energy is greater or less than zero. You see this more in Player Puzzles than Challenges or Labs. And the local allocation of free energy in a branch or can affect the stability of stacks and loops in that branch.

But free energy is *not* the be all and end all of stability. You often see new players wanting to get the Free Energy as low as possible (I did for a while), but this can mean using mostly GC pairs which are prone to mismatch, and hence *decrease* the overall stability. In the labs this shows up as messy dot-plots.

Think of it this way: for a given base sequence [ACGU…], the most stable shape [.((…))…] is the one with the lowest free energy. But for a given (complex) *shape* the most stable *sequence* is not always the one with the lowest free-energy, rather a “Goldilocks” sequence: not to much free energy, not too little free energy, but just right!

Thanks jandersonlee, that helps.

Maybe you could help with something even more basic… Sometimes I need a ‘branch’ of bonded pairs to go in one direction, but when I click over to Natural mode, the bonded pairs that should hold the shape of the loop switch and bond to other nucleotides. If I’m already using GC pairs to hold the loop, and supporting them with adjacent G’s, what else can I do to make the bonds I create stick when I switch to natural mode? Does this part of the game make sense if you understand how energy factors into the equation?

In one sense energy does play a factor, because the game is preferring one shape over another due to overall energy. The energy is lower in the shape that is picked as the *natural mode*.

That said, the solution can vary. You sometimes need to do what you can to *discourage* the natural mode mismatched shape rather than *encourage* the desired (target) shape (via boost points and stronger bonds etc.). This can often be done by changing one of the bases that part of the mismatch is built on.

One technique is to use ctrl-click to mark bases that are mismatched in natural mode, then flip to target mode and see where they end up. If part of a mismatched pair ends up in a target mode pair elsewhere it is sometimes as easy as flipping that pair to discourage the mismatch for example. If part of a mismatched pair ends up as an unbonded base in a loop, then sometimes changing that base to a “blocking point” (e.g. A->U or A->C) can work. Sometimes changing one pair involved in a mismatch can help such as from from CG to AU or vice versa.

But ultimately, the target mode has to have lower energy than any alternatives.

Would it help to learn successful patterns of base pair placement if we could save solved puzzles with energy amounts assigned to loops for comparison with other solved puzzles of a series? I thought of this but it would be time consuming to make diagrams and I am not sure of its value.

I am also not sure of its value. As jandersonlee explained, it’s more about a comparison of target and natural energy than driving toward any particular energy value

I also would like to know the significance of the score given when a puzzle is completed, and if there are strategies beyond simply finding a stable arrangement to complete the puzzle. I imagine there are numerous solutions to each puzzle, and there must be ways to increase one’s score (and perhaps the effectiveness of the molecule?) Just shoot for the lowest energy score?

i have no idea

I think that our task in the game is just to create different possible combinations for the same molecule.

The concept of (Gibbs) Free Energy can be very confusing. Gibbs invented the concept to provide a criterion to decide whether a process in a system of interest (like an RNA molecule in solution) that can exchange energy with its environment is spontaneous under conditions of constant Pressure § and Temperature (T).
According to the 2nd Law of Thermodynamics, for ANY process to be spontaneous, the change in entropy of the “universe” (defined as the system AND its surroundings) must be > 0. Entropy is measured in units of Energy/Temperature (e.g. Joules/Kelvin or Joules/(mole x Kelvin)). The entropy changes of the system and of the surroundings can be computed separately and added together to give the total entropy of the universe. Since the surroundings are much larger than the system, their temperature does not change during the process and so the entropy change can be calculated by dividing the thermal energy (represented by the “enthalpy” change) transferred between the system and the environment by the absolute temperature. The more energy is transferred to the environment, the more its energy and therefore its entropy increases. When an RNA folds to form a structure it generally forms a lower energy state, and therefore releases energy to the environment (by conservation of energy = 1st Law of Thermodynamics) increasing its entropy. Concomitantly, RNA folding often releases ions and solvent, increasing the entropy of the solution but at the same time decreasing the entropy of the RNA chain itself, so the entropy of the RNA itself can be lower after folding. The important thing is that the overall entropy (of the “universe”) increase for the folding to be spontaneous. Which term dominates is very challenging to calculate and depends on the particular RNA.

Gibbs simply multiplied the expression for the total entropy by -(T) to produce a quantity with units of energy that he called “Free energy.” He gave it this name because at conditions of constant pressure and temperature it also corresponds to the maximum amount of energy released by a process that can be converted to useful work. But that is another discussion.