I have written before, about placing a lot of nucleotides in the loopring, probably not were a very good idea. It was generally safer to leave them yellow, as they were. Unless the added nucleotides were in colors and position where they were previously known to function well. Ding came with examples of working ones in Jeehyung’s post, Tell us about your EteRNA lab algorithms!
Now our new advance energy mode shows us what is going on. Placing nucleotide G,C, U beside the GC-pairs, changes the energy level inside the multiloop
I tried this operation on both Mat’s Branches V1 (90%) and his Branches V2. (95%) with the exact same result. The numbers in the spreadsheet also explain why blue is the absolute worst choise when it comes to nucleotides in multiloop rings. Something some of us already have noticed. Here is the link to the spreadsheet:
This finding is strongly related to AnticNoise’s discovery of energy gaps, to the samedirectional rule for GC-pairs in multiloops and even distribution of energy.
Turning the GC-pairs in the opposite to usual direction (red nucleotide to the right) makes the energy level go towards positive numbers.
Placing G, C and U’s in the multiloop, makes the energy level go towards positive numbers.
As the stacks/arms have energy levels with high negative numbers, turning GC-pairs in multiloops and adding other nucleotides in the ring of the multiloop, creates a bigger energy gap between the parts of the designs, which if too big, makes the RNA structure break.
I think though, that in some cases knowing how this work, will be useful and a few different turning GC-pairs or added ringnucleotides might be even helpful.
I think when we understand more about the nature of RNA, we may learn to see the direction of GC-pairs in multiloops and the placing of nucleotides other than A’s inside the multiloop, also as two different handles which can be used to change the energylevel inside a multiloop according to what we need to balance the energy in the design and make it stable enough to fold correctly.
But the general rule is, that there is a right and wrong direction for placing of GC-pairs in multiloops. And that adding of ring nucleotides other than A’s, usually means trouble. Adding both othercolored ring nucleotides and turning GC-pairs the wrong way, doubles the trouble.