I am trying to bind C in A*B/C INC at 300Nm but when I do it also binds at 100Nm. When weakened it won’t bind in either state. Why is this so?, and what is the fix?
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Speculating (hopefully someone more experienced will chime in) it’s because while weakening the binding to C you’re also weakening the A/B binding: you want to weaken the bit that binds C only. Easier said than done I know.
The energies of the two desired states must get close in the range of 0 to 1.36 kcal. When you change the design your change might change the energies of both states by some value.
The trick is to change it in such a way that the difference in the energies of the two states shift (rather than stay the same) in the direction of this 0 to 1.36 interval. In your case your change either affects both desired states energies in the same way or the shift is not enough.
If you post your raw design I might be able to give you an example.
Make more UG or GU pairs they are for switch they gives a little bit less negative energy in one stage but at other stages you can bind with AU UA or CG GC pairs.
At start and at end of oligo put extra nucleobase it gives negative energy (better connections)
A base gives max negative energy (max stable)
C less
G less
U minimum negative energy (max unstable)