I am new to this community. Just created the account. I would like to ask the best players: What do you look at when you vote for a RNA molecule? It’s not just it’s free energy, right?
Ok, I answered your question first in chat and saw this later. I’m going to answer here anyway, as somebody else might want to know too.
As you totally correct guessed, energy is not the only thing we look for. We look for things like if there is a pattern in % of GC-pairs, AU-pairs and GU-pairs. In which range do a lab design do well? Same with melt point and dot plot.
When we get results back from lab, we look at if there are things that seperate the correct folding designs from the ones that do not fold well. If there are patterns occouring in bad folding designs that are not present in the high scoring ones. So vote for designs that looks like winners, with absence of the patterns from bad scoring ones.
I have described some of the patterns I see for single shape lab, in My strategy guide to the lab
(Note this will not necessarily work in the switch lab. It is mainly an exampel on what kind of patterns you can look for)
Now we are working on shapeshifting RNA in the switch lab. It is still quite new and we are not yet too sure about what works.
Only advice for now is that someone mentioned that energy difference between the two shapes seemed to matter, I checked myself afterwards. My advice is try keep the energy difference between the two shapes between 1 and 3, because the designs with lower or higher difference seems to score low. So take note on this when you design yourself or vote for a design.
You can find more lab introduction in A comprehensive guide to EteRNA.
Early in the lab rounds I tend to look for different things than later, and as time/rounds goes on I may have different ideas/hypotheses I’m playing with. Sometimes I may have different conflicting hypotheses to test out, so I may vote for several designs that are vastly different. We don’t yet know what strategies will work for switches, so a sampling of different designs is useful in early rounds.
In general, I look for designs that have a moderate number of GC bonds in both target shapes, between 1/3 and 3/4. Too few is a “cub scout” and too many is a “Christmas Tree”. Neither tends to do well in the lab. This also tends to rule out designs that have extremely low free energy (which often don’t work well by the way). GCs also tend to do better when placed next to a loop for short stacks.
I also look for designs that seem like they might be free from (or contain very few) sequences that are prone to mismatches seen in other switch lab results. That includes CC (mismatch with GG), UGU (which can mismatch with the fixed ACAs in the hook), GUG (which sometimes mismatches with a fixed CAAC), and UUC plus UCU (which can mismatch with a GGA in the hook or aptamer loop). I’m also playing with the idea of minimizing the use of GU and UG sequences and UG bonds as a test of a one of my own hypotheses; but that would just be in one or two designs - not in general.
For the current switch lab, I try to find two or three designs that have fairly clean dot plots with a strong suggestion of both target shapes in the dot plot and vote for those. I don’t tend to look at the melt plot to closely myself - though some people do.
As Eli suggests I mostly tend to vote for switch designs with a narrower range of free energy difference (about 1.5 to 3.0 for me) between the two target shapes (not counting the bonus). I might occasionally vote for one or two that have less (0.8 to 1.5) if and only if there are other positive aspects to the design.
In terms of other personal “pet projects/ideas”, right now I’m playing with the idea that shapes fold more easily from the end-loops back towards the hook, so I try to vote for a few designs that seem like the energy of the end-loop is a close match to the energy of the closing stack cell for the bound form and that have a stronger stack closure for the end loop in the unbound target. The hypothesis is that this will help the shapes to form more easily.
I’m also looking at the free energy of intermediate forms that the RNA might encounter in refolding from one target shape to another and voting for a few designs that pass through lower energy intermediate forms. That’s not something you can tell directly by looking at the design, but I will often add a comment to a design that I vote on which seems to meet that criteria.
In later rounds I often reserve more votes for designs that have made small changes to designs that have been already synthesized and scored well, especially if it looks like the change may have addressed some problem area in the design.
If you can make the time, look at some of the better scoring results from the previous switch lab and see where they seemed to go wrong (i.e. mismatch); that may give you some clues on what to try and avoid.
All in all, try and come up with a few *different* ideas on what you think might help or hurt a switch design and give votes to designs that you might expect to do better based on one or more of those ideas. Over time we will hopefully get a better idea of what really seems to work.