Questions about the new reporter RNA

General Science Discussion
1

I have a two questions now; more may well come up.

  1. Is the length of the reporter sequence highly constrained by the chemistry for fluorescence?  I ask because if we believe in the NuPACK energy model, there isn’t much room for choice in a matching sequence of the switch that keeps it bound.  This does not seem to be a problem with the initial TB input molecule and the single input case, but it could be a cause for concern when we get to multiple, novel, might be with some other input RNA(s).  Having a few more bases in the reporter would give us more flexibility for balancing the energies between states

  2. How is the reporter RNA linked to the fluorescent protein?  In particular, will the protein fluoresce when the reporter RNA is bound to the switch RNA? Or not bound? 

2 Likes
2

Apologies for the gibberish sentence.  It is supposed to be

This does not seem to be a problem with the initial TB input molecule and the single input case, but it could be a cause for concern when we get to multiple, novel, input RNA(s).  

But for some reason I can’t edit it.

3

Thanks for the questions.

  1. The length of the reporter is not really constrained by the chemistry for fluorescence. The fluorophore will be attached to the end and the RNA we choose can be of different lengths. Initially we chose a rather short RNA that is expected to come on and off on the time scale of our experiment. For switches with RNA inputs and outputs, strand displacement means that we can probably use longer RNA molecules and we hope to incorporate this for future rounds.

  2. For now, we will use either a fluorescence RNA molecule OR a protein. We may even use multiple fluorescence RNAs in the future but that’s for later.

2 Likes
4

So the reporter RNA fluoresces all by itself when it isn’t bound to anything?  I didn’t know any RNA did that.

Or maybe I didn’t understand, since the first answer said the fluorophore would be attached to the end of the RNA.

In any case, will the fluorescence be observed when the reporter is bound or unbound to the switch RNA?

5

For the “output” RNA molecules we will chemically attach a fluorophore such as Cy3 that provides the fluorescence signal. Normal RNA would not fluoresce under these conditions.

6

Thanks.  I think I understand now.  But to be sure, let me check.  In the RNA In/Out puzzle, a successful solution will release the reporter RNA/fluorophore molecule, which will be washed away. So a cluster that is successful will fluoresce in state 1 (because the fluorophore is bound to the cluster) and not fluoresce in the second state.  Right?

1 Like
7

Yes, you’re absolutely right. For a successful switch one of the states will hopefully fluoresce and the other will not.

In practice the fluorescent signal may be concentration dependent but the goal is to see different signals in the two states.

1 Like