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Base pairing

Hydrogen bonding in Watson-Crick base pairs.

GC pairs (or CG pairs) are base pairs that form between guanine and cytosine. GC pairs are stronger than AU or GU pairs.

Most successful lab designs contain between 50-70% GC pairs. Designs that contain an excessive amount of GC pairs are difficult to synthesize and prone to misfolding. These designs are known as "christmas trees"

Why don't I want to use all GC pairs in my lab design?Edit

Below is a post and an excellent example from Chris Cunningham in his excellent thread 'Why Wouldn't I Want to Use All G-C Bonds?'

Folding Paper Suppose you had a piece of normal-sized paper, and you had some glue. Put glue all around the borders of the paper, and fold the piece of paper in half. You want the paper to stay glued together and perfectly folded in half -- no bumps or creases.

  • The question: why wouldn't you use the strongest glue possible?
  • The answer: because while you were folding it, every little mistake would be permanently fixed in place, so you wouldn't actually get the right shape. Actually you don't want the strongest glue, so that you can fold the paper in half and iron out the creases and bubbles once you have the basic shape down.

Folding RNA

So now suppose you have a strand of RNA, and you get to choose its bases so that it folds exactly in half. You choose the bases, and you have the option of either choosing all G-U Bonds (weak glue) or all G-C bonds (strong glue).

  • The question: why wouldn't you use the strongest bonds possible?
  • The answer: because while it is folding in half with G-C bonds, every little mistake would be permanently fixed in place, so in the end you probably wouldn't get the right shape. Actually you don't want the strongest bonds, so that you can fold the RNA in half and it will be able to iron out the creases an bubbles once it gets the basic shape down.

How can I use this in the lab?

The basic thing you can take away from this idea is that the most-negative-free-energy design is not the best one. That design necessarily uses a lot of G's and C's ("very strong glue"), so while the RNA is folding and it inevitably folds a little bit wrong in some spot, it will have trouble unsticking at that spot in order to get to the shape that is actually the most stable.

So, very negative free energy is bad?

No. If your free energy isn't very negative, then it is like not using glue at all -- your RNA won't stay in place even if it finds the right shape. The correct amount of free energy to use in the lab is likely a balance between making it sticky enough to stay together but not so sticky that it gets stuck the wrong way. "" Submissions to the lab that have a 100% GC content will not be selected for synthesis, no matter how many votes they recieve.

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