Identifying Regions Important For Secondary Structure Formation In The Entire Length Of A 3'Utr

Question

[ancient_learner]

I am having some problems cloning a big 3'UTR for luciferase assays. This particular 3'UTR is 3.2kbs in length, and I cannot clone it after several attempts. Iam planning to clone a small fragment of it (obviously containing the predicted binding site for my miR of interest).

Infact 3 of individual mirna BS (binding sites) are located within 1st kb of 3'UTR of that particular gene. I have doubts about this, because I am thinking that perhaps these particular miR binding sites could be, for example, functional only when some secondary structure of the 3'UTR forms (in the presence of other miR binding sites of the 3UTR). Which would mean that if i leave them out, I might not be recapitulating the true miR-mRNA effect. so if there is any way to identifying regions important for secondary structure formation, site accebility etc in the entire length of a 3'UTR i hope it would be nice. i can clone only that region say 1-1.5kb including BS I found. otherwise any other suggestions are also welcome on how to solve this problem

Answer 1

[Devon Ryan]

Interesting issue, I'm a little surprised that you've run into such issues with the subcloning. If you really do have to resort to just using a portion of the UTR, you can predict secondary structure with the RNAfold program from the Vienna RNA package (there's a web version, which makes things convenient). I would suggest that you continue trying to subclone the full UTR, just to avoid this issue. You might ask around for help from others, there are a lot of PCR tricks out there.