Hi Keteryne Silva

I'm also looking for the same thing. Hope members will provide us some info.

Cheers!!!

Hi Keteryne Silva ,

You could try with targetscan, it gives you a visualization of how many nucleotides of your sequence are binding with the miRNA.

That should give you an idea of where to place the mutations if you want to destabilize it, but variations in the rest of the sequence may also have an effect, so you may want to test a few constructs.

All the best,

Mauro

You do not have to use modified primer. Instead, you have to mutate the seed region of the 3'UTR site in the Luciferase vector. This can easily be done utilizing a 3'UTR mutation Kit (e.g. Q5® Site-Directed Mutagenesis Kit, NEB). In the guideline of the manual, all questions regarding primer design etc. are mentioned and all prosecces are described. Possibly, the following Research Gate discussion may help you (https://www.researchgate.net/post/How_should_I_mutate_the_3UTR_of_microRNA_binding_sites).

Alternative, see for ref. J Hypertens. 2016 Feb;34(2):323-31. doi: 10.1097/HJH.0000000000000799. or other paper dealing with verification of miRNA-specific binding to a target 3'URT (see PubMed.gov).

Mauro Lago Docampo I already have the sequence where the miRNA should bind the gene (targetscan). My question is if I should design another primer with a random mutation in this site or if there is a specific tool that will insert a specific mutation there.

Thanks!