I am performing miRNA loss-of-function study in cells and using a reporter assay to validate miRNA inhibition. So, I am planning on cloning the entire 3’-UTR sequence of our miRNA target downstream of a reporter gene. Along with this WT clone, I would like to include a negative control clone that contains a mutated sequence cloned in the place of miRNA target sequence. Would altering a single base in the miRNA seed region be enough to keep the miRNA from binding to the target sequence? or should I consider altering the entire seed region? Also, the miRNA has more than one seed region in 3’-UTR of a target. Any thoughts/suggestions would be greatly appreciated.
Dear Ramana Vaka,
I agree with Reynaldo Garcia
that depending on the length of the 3'UTR, some noise by other factos/ miRNAs might disturb your experiment. However, if you choose a very short sequence with only a couple of bases flanking the binding site, the experiment becomes quite artificial. So if you have like three miRNA binding sites within ~1000 bp of UTR I would give it a go and see if a miRNA mimic downregulates the reporter. Concerning the mutation, you might want to keep in mind that changing only one base might result in some wobble base pairing and could even enhance downregulation. So maybe try to change about 1/3 of the seed region and check that you do not create a novel target sequence of another miRNA that is highly expressed in your cell system.Good luck
Boris
Thank you Reynaldo Garcia
& Boris Schmitz for your suggestions.Before I wanted to go with target 1 (2 seed sequences in 3' UTR, length 2170 bp). However, based on both target prediction (target scan & miRDB) and experimental evidence, I have another target 2 (1 seed sequence in 3' UTR, length 839 bp). Since target 2’s 3' UTR is relatively short and has only 1 seed sequence, I think it makes to sense to go with target 2, do you all agree? The reason I wanted to clone the entire 3' UTR sequence is to preserve RNA secondary structure (if any, might affect its binding with miRNA)
Regarding mutation-Splicing-by-overlap extension PCR mutagenesis seems like a great method for this purpose. Creating a novel seed sequence for a different miRNA has been my concern as well. Is there a resource you aware of to look up miRNA(s) by searching with seed sequence?
Thank you again.
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