How to overexpress star miRNA (miRNA*) from pre-miRNA construct - (Oct/18/2009 )

How to overexpress star miRNA (miRNA*) from pre-miRNA construct

I am working on miRNA* field. I plan to clone the pre-miRNA (about 500 bps with flank sequence) into vectors, then express mature miRNA in which the miRNA* is only by product. But, how to construct the pre miRNA vector in which the MAIN PRODUCT is miRNA*, not mature miRNA. I have no any clue on it and no papers on it.

Thanks in advance

My two cents.............

My suggestion is using antisense inhibitor to block the dominant counter-mir from your pri-miRNA expression. But by doing this, I still don't think the mir* is "OVEREXPRESSED" (if this is what you want?), the mir*just becomes more dominant. The similar way is to change the C on the counter-mir to U (T) to eliminate its function but keep the base-pair.

You can also try to make siRNA-like double-stranded RNA with GC base pairing in one end to favor the miRNA machinery to take the mir* sequence, however, DICER might chop your "siRNA" into 19-mer and I think that's the drawback. Alternatively, if you can change the base-pairing in the pre-miRNA to make mir* being favored, then it might work.

Comments..............?

You could also try a miRNA sponge approach to sequester mature miRNA but leave miRNA* unaffected.

Thanks a lot. To functional screening, in your post, you said maybe to change the basepair in pre miRNA to make the *miRNA as favorite product. What is the principle I should use to change the basepaire? Are there any papers to discuss about this problem?


Thanks again in advance

veer2 on Oct 25 2009, 10:19 PM said: