Synthetic Biology:BioBricks/mRNA design rules

Problem: How do you insulate parts so that mRNA structure doesn't unpredictably effect performance when they are combined.

• Austin Che: Perhaps we should limit to specific cases where we care about. For example, RBS and coding sequence seems to be the main junction we care about and inside this junction, we can't really insert any buffering sequence. Transcriptional terminators and other RNA signals are another category of things we care about.

1. Buffer regions
   • add flanking regions of "meaningless" sequence that will buffer parts from each other and reduce likelihood of secondary structure at the part junction.
   • how long should this sequence be? should it have no structure?
   • Austin Che: One of the papers from last lunch I had picked had shown that RNAs fold locally within about 50 nt and that going any further out actually decreases predictive ability. "no structure" is a structure. It seems you want a buffer region that's the most unlikely to occur in a normal sequence. If normal sequences have "no structure" then "no structure" buffer may pair with the "no structure" sequence. Conversely if the normal sequence has lots of structure, making a buffer with high structure may cause high structure between the buffer and target sequence. Thus, it seems that it depends on what we expect our target sequence to look like.
2. Automatic checker
   • look at the secondary structure at the part junction and predict whether it's going to be a problem, if it is put up a flag for the user.
   • how would we do this exactly -- Sfold the entire part and then looks at the structure of the junction? what are our cutoffs likelihoods on the folding, etc? If we ran every BB part against every other one what fraction end up with some structure accorss junctions?
   • Austin Che:It's nice in principle but not clear whether this is any use in practice. I'll wager you'll always find some structure across the junction, probably from really far apart sequences which may not get the chance to actually come together in the cell.
3. others?
   • Austin Che:You should take a look at this paper from Burge's lab. I didn't add it to list of articles for last lunch because it isn't about the accuracy of computational prediction but it is very interesting in that it finds that mRNAs have more local structure than non-coding regions. They don't really explain it satisfactorily (in my opinion) but it could be that increased structure in coding regions decreases the chance that surrounding sequences can affect its structure. So we can perhaps increase insulation by choosing codons that increase structure in coding regions (or use my algorithm of finding the codons most immune to insertion of random flanking sequences).

  Katz L and Burge CB. Widespread selection for local RNA secondary
  structure in coding regions of bacterial genes. Genome Res 2003
  Sep; 13(9) 2042-51. doi:10.1101/gr.1257503 pmid:12952875