This is a thread for discussing the analysis of the new
riboswitches that uses different aptamers as inputs and MS2 or kissing loops as
reporters. The aptamers in question bind tryptophan, theophylline or arginine, respectively, which presented new challenges compared with earlier puzzles using an aptamer for FMN. Looking forward to the discussion.
There is also a separate thread for the kissing loop puzzles: https://getsatisfaction.com/eternagame/topics/kissing-loop-riboswitches
The puzzles were posted as two separate Eterna labs:
Single-input switches, revisited: http://www.eternagame.org/web/lab/7559749/
Single-input switches, revisited (with RNA kissing loop probes): http://www.eternagame.org/web/lab/7630523/
Initial data are available (both kissing loops and MS2):
Results R107 - Data: Fusion Table + Switch Graphs
Heads up. If you can't find a design by its designID in the lab where you think it belongs, there is a reason. The data are from two bigger and seperate labs, but some of the sublabs have the exact same names.
There is an extra column in this data collection called Puzzle_Type. Use this one as a filter and that way you can get the kissing loops labs split from the ones with MS2.
The MS2 hairpin works better than the kissing loop hairpin for all but tree labs
(Same state - Argenine B, Same state - Tryptophan A, Exclusion - Argenine A. In Same state - Argenine A, there is a draw between MS2 and the kissing loop hairpin.
Transfer of the FMN/MS2 riboswitch blueprint
The riboswitch blueprint as seen in the FMN/MS2 riboswitches is also present in the labs that holds new aptamers and one of the lab rounds holds a kissing loop hairpin (K4) instead of a MS2.
This can be seen in the following things
The puzzles of the B type (that has the aptamer optimally oriented towards the switch area) generally can be solved having the switching elements inside a switch bubble.
The best Same state B type designs tends to have the MS2/K4 distanced to the aptamer - as were the case for the FMN/MS2 riboswitches
Main part of the best Exclusion B type designs have the MS2/K4 distanced to the aptamer as were the case for the FMN/MS2 riboswitches.
There are more exceptions though, which I find interesting. However these exceptions do turn up in particular in labs where we have a hard time hitting a decent score. Something which were also the case in some FMN/MS2 Exclusion labs. But in those Exclusion FMN/MS2 labs where we had trouble making winners, the FMN aptamer was not optimal orientated and not optimal placed. (more of an A type placement).
In the labs where it seemed hard to get winners, it was seen in exclusion designs that the MS2 was moved further from the aptamer. Which is akin to solves the riboswitch labs, Exclusion 5 and 6. Those were also more likely to be solved in an open ended style due to the aptamer not turning in the optimal direction.
The aptamer is likely turning the wrong way in these labs
As I mentioned in my earlier Kissing loop analysis the following labs probably have their aptamer oriented the wrong way in the following A type puzzles of both Exclusion and Same state kind in both the K4 and MS2 lab type. :
KDON and low scores
There is an overall high KDON for the main part of the labs.
High KDON means that it is hard for the aptamers to bind the molecule in state 1. It seems hard to get a low enough KDON for the aptamer, to achieve a good score in a lot of the labs.
Average KDON and KDOFF for Kissing Loop riboswitches
Average KDON and KDOFF for MS2 riboswitches
The B puzzle types were more likely to be solvable having the switching parts locked in to a switch bubble where the switch elements are brought close together.
This conformation was also present in the most successful of the MS2/FMN riboswitches. The switch bubble pattern showed itself in the FMN/MS2 puzzles when
The MS2 was put in between the FMN sequence (akin to the B type puzzles)
The FMN was orientated in its favored direction
This happened exclusion style labs too if there were enough sequence length to make a switch bubble and the FMN was had its favored orientation towards the switching area.
The A puzzle labs were more likely to be solved in an open ended style.
This is likely partially due to not all the aptamers having the optimal orientation. I will expect more switch bubble style solves if we saw the round rerun with the A puzzle types having their aptamer orientation reversed. (Except for tryptophan which is turning the exact same way in relation to the switch area for the A and B puzzle types. It is likely turning in the primary orientation already.)
Theophylline goes both ways
One thing I find really interesting is that Theophylline do not seem nearly as picky about what way it is orientated as the FMN aptamer. There are winning designs in 3 of 4 theophylline labs and a near winning score for the last one. Despite the aptamer being inverted for the A puzzle types and the designs being solved in an open ended style.
We have scores of 100 in both the Theophylline in both the A and B puzzle type, despite the orientation of the aptamer in relation to the switching area is not the same. Actually I can’t even say that. Because the Theophylline Same state A lab have a winner in an open ended style solve with an inverted aptamer. So despite the aptamer turns the opposite way to the B type puzzle, the aptamer is actually orientated the same way in relation to the switching area - which this time just is out. Not inwards inside a switch bubble. This is new and very interesting.
I have taken notice that longer stretches of G and U seems to badly effect the score.
So far I have gone through all the theophylline/MS2 lab data and my suspision has only been confirmed. Designs with longer stretches of GU's disproportionally ends up with lower scores + raised fold change error.
However there are some good scoring designs that do also hold a stretch of G and U, but I think position matters. It may be worse if it is close to or inside the switch elements. Also not all combinations of G's and U's seem equally bad.
I suspect this is why that we have gotten less good results on the Kissing loop labs, as the Kissing Loop itself holds this G and U pattern. In other words, I think we will get better results with kissing loops, if we just pick one with another - and more switch like sequence.
Discussion with Omei from 27 Feb 2018
eli [12:13 AM]
In most of the labs it looks like MS2 performs better than KL
But then again, KL doesn't have MS2 complementary bases, as did the FMN aptamers and other of the aptamers
So less luck for direct pairing up for shutoff
omei [12:16 AM]
It seems like the KL locked few enough bases that additional stem was required. Why couldn't we create whatever complementary bases we wanted as part of that stem? (edited)
eli [12:21 AM]
Ok, we could and we did.
eli [12:21 AM]
uploaded this image: image.png
eli [12:21 AM]
But there is something else different about this switching hairpin than MS2
It holds excess G and U.
I have regularly seen excess G and U in designs that don't switch well.
The kissing loop labs generally don't reach high fold change
I'm not certain they are to blame, just wondering
omei [12:25 AM]
Do you consider the design you pasted to have excess G and U?
FWIW, Joanne is inclined to believe that the KL aptamer doesn't bind as well as was described in the paper he got it from, at least not under our experimental conditions.
eli [12:30 AM]
No, just the kissing loop itself.
I recall earlier labs where I tried a GU rich turnoff sequence instead of a CU rich one. And it tended to fail miserable.