I was at writing report on EternaBot to see if I could spot some things that could improve its score, and while I was at it, I decided to take a look at the player designs too. Basically with the same aim. :) We have done good, and I think we can do even better. So here are some things I think are worth looking out for.
I have summed up, what I think was a problem for each lab type. I hope this will be a help for understanding what can be a problem in a particular type of lab design and why.
A switch design isn’t just a switch design. Switches have personalities. They want specific things, just dependent on their structure. Also they don’t come as equally hard to solve. The easy labs takes a certain solving style that won’t work on the hard labs, and similar the hard labs takes a different solving style that won’t work on the easy labs.
I think if we use this knowledge, we already have very strong cards on the hand to tell what a particular FMN/MS2 switch needs, just from what its structure is. I strongly suspect that this will be applicable to other types of switches too.
Exclusion 2, 3 & Brourd’s mod of Exclusion 4 - Easier labs
Here I list what I think gives bad scores for this kind of easy labs.
I have included Brourd’s mod of Exclusion 4 in this series, as it does what the easier labs, Exclusion 2 and 3 does. Allow the switch to get locked away inside itself. I will just mention on which cases it sticks apart from the other 2.
End tails not tied with each other = unstable aptamer end
End tails dangling but not involved in anything
Aptamer not locked from one end
Moving: Too much of the design switching - not partial enough switch
Attempts to bind up way too much of the MS2
Way too long aptamer gates
Way too long MS2 turnoff sequence
No MS2 turnoff sequence next to the MS2 sequence
Turnoff sequence not pyrimidine enough. :)
Turnoff sequence length too short or too long. Needed: 4-5 bases with a little more to Exclusion 2, because this lab is harder due to the left positioning of the MS2 turnoff. But turnoffs that get more than 6 bases needs splitting - else I suspect they make the MS2 stay turned off and not capable of switching fast.
No static stem made of excess bases
Not needed for Brourd’s mod of Exclusion 4, as there were no excess bases. Which is actually kind of interesting given that we have been easier able to make higher scores in the other two labs that allowed space for excess bases. However this Exclusion 4 lab still do have the disadvantage of having the MS2 and FMN 1 base apart, which Exclusion 2 and 3 don’t. So I can’t say which is which.
No Multiloops, many of the lower scoring designs have no multiloop. It is the natural consequence if the tails are not knotted with each other.
No salish hinge (3-4 base sections of mainly A bases spiced with an occasional U or C, located in the multiloop in the switching area. Should be after the aptamer gate strand that does not have the the MS2 next to the aptamer.
Exclusion 2 needs the Salish hinge less than Exclusion 3, namely because Exclusion 2 needs a stronger MS2 turnoff. But I’m guessing it will benefit the results, should we have a few extra bases space for getting it in.
Turnoff sequence not pyrimidine enough.
Too long stems, 6+ and above is too long.
Too many switching stems. Exclusion 2 lab is harder than Exclusion 3. This lab benefits from 2-3 switching stems, normally I will only recommend 2 (Counting MS2 as 1). The reason for the extra needed stem, is that the MS2 needs some more help with getting turned off. Making the MS2 turnoff too long, is not working as well. I suspect it prevents the design from jumping states as quickly. It get too stable in one state over another.
Too much GU’s. Fairly easy riboswitch labs mostly takes a % of 0-5 and sometimes up to 10% above that and it is not helping. Especially not if in the switching area. You may get easier away with it in static stems.
For some reasons Brourd’s mod of Exclusion 4 takes a little more GU than the more average easy switch lab. Trends are that the harder the lab gets, the more GU. I consider this lab harder than both Exclusion 3 and 2. One of the reasons for that is that it have introduced 1 base distance between the MS2 and FMN, just as the Exclusion 1 and 4 lab has. But I think this small distance already starts makes the lab goes harder, despite it has long enough tails to knot. Fairly easy riboswitch labs takes a GU of 0-5% and sometimes up to 10%. This lab can go to 10%.
Too much GC’s or too little. Generally switching stems should have one 1 GC pair as a minimum, but they don’t like too much more either. Often 2 is the maximum, unless there are special circumstances that does that the lab needs more. Basically how many GC’s you need per stem, depends on what you want a switching stem to do. If you want to turn off the MS2, you will tend to need in the high range of GC’s. To counter the MS2 hairpin forming, as it really want to fold. At least in Exclusion labs. So for the turnoff sequence you can regularly away with more than 2 GC pairs. Still it depends on the general hardness of the lab. Exclusion 2 is harder than Exclusion 3, because it has its MS2 turnoff sequence to the left, where it has weaker effect on the MS2, than if it was to the right. So to solve Exclusion 2 more GC pairs (forming between the MS2 turnoff and the MS2 sequence) are needed for the MS2 turnoff.
Exclusion 1 & 4 - Harder labs
Here I list what I think gives bad scores for this particular kind of lab. and what elements are missing.
MS2 not caught from both ends. JR had a top scoring exception in Exclusion 1.
Aptamer not locked from one end. Although open ended designs can contain a loop around 3 bases away from the “static” end of the aptamer, as Omei noticed.
Attempting to tie up the tails as if it is an Exclusion 2 and 3 lab, is a bad idea - the tails are too short to hold the pressure from the rest of the design attempting to switch. But I had to try. :)
Too long end tails dangling but not involved in anything
Too much of the design switching - not partial enough.
Too static - trying to solve the design with too few parts switching. Absolutely not working. But I just had to try. :)
Multiloops - happens when the tails are paired and these are not helping. As I suspect it is not holding.
No MS2 turnoff sequence right before the MS2 sequence
Turnoff sequence not pyrimidine enough. (Exclusion 4, not the case in Exclusion 1)
Aptamer gates - too short. Typical length 3-7 bases - longer is probably due to the lab being hard to get to switch its MS2 as it is not bound up from both ends.
Too many switching stems. Open ended switches tends to needs fewer switching stems compared to easy to solve labs and pressured labs.
Too much GU and too little. Open ended labs tend to allow a bit more GU’s. A healthy range is typically around 5-10%
Too much GC’s or too little. Generally switching stems should have one 1 GC pair, but they don’t like too much more. MS2 is the exception. So like this, you should usually have one GC in a switching stem, if it is short you may get away with 2 GC pairs. Though when countering the MS2 hairpin which really want to fold with it self you can get away with more than 2 GC pairs for the turnoff sequence.
Exclusion 5 & 6 - Hardest labs
I list what I think gives bad scores for this particular kind of lab.
End tails tied with each other = too stable design and aptamer end. But I had to try. :) Result = Bad
Lack of tail dangles - but hard full moving labs seems to like them - contrary to partial moving switches
Aptamer locked from one end. (Exclusion 6 does allow that in some of the higher scoring designs though). These labs tends to need to go towards full moving. Having too many static stems are counterproductive to be able to get a solve. Not that it did not stop me from trying it out. :) Result = Bad.
Partial solving style is generally not working - these switches needs to move. :) But I still had to try. :) Result = bad.
(Exclusion 5 needs to move more than Exclusion 6)
Multiloops are not helpful for making full moving switches move. They add way too much stability. :) Of cause I had to try. Result = Bad.
Static stem - should be banned. They prevent the switch from reaching its full moving potential. I did try. Result = Bad.
Not enough turnoff sequences, more are beneficial as one typically can’t reach both switching elements due to switch element distance. Often both FMN sequences needs to be caught by turnoff sequences. I did try with a single one. Result = Bad.
There needs to be at least 2 and sometimes 3 turnoff sequences. One for the MS2 and one for each of the aptamer sequences. The switching elements in a full moving switch does not always want to share turnoff sequence.
Exclusion 6 also benefits from a slightly more purine MS2 turnoff, compared to the more usual pyrimidine turnoff sequences. Actually it appears that this exact lab doesn’t mind the longer stretches of G’s and U’s, compared to the easier labs. The GU electronegativity and their not wanting to bind properties in especially switch designs, may be an advantage here, like oil reducing friction. To get the full design to move for making the sequence go unstuck and switching. (Penalize GU heavy regions)
Too much and too little GU’s. The harder the switch, the more help it needs to get moving. Typically takes around 5-10% GU’s and sometimes more. Hard labs like these tend to accept more GU’s than the easier labs.
Too little GC pairs. Exclusion 6 take a higher amount of GC than usual in the switching area. (which is the whole puzzle) Exclusion 5 on the other hand takes more AU. I’m beginning to think that these two designs with their switching too apart, are quite different, despite sharing a lot of tendencies.
Extra comments on Exclusion 5 & 6
Exclusion 5, tends toward being first half of the RNA pairing up with the last half and then a small slide. Example of jandersonlee’s high scoring mod of Malcolm.
Score 85%, cluster count 53, fold change 7.55
Exclusion 5 lab benefits from having 2-3 internal loops in either state.
Exclusion 6, too much of first half of the RNA pairing up with second half of the RNA. Needs to be split in two sections, that switch with each other.
Jandersonlee’s winning mod of ViennaUTC in Exclusion 6.
Score 96%, cluster count 30, fold change 18.66