I have to do a Biotinylated RNA pull-down assay to verify the ability of two RNA binding proteins to bind RNA of interest. I’m working on designing the oligos. I wonder it is better to add biotin at 3’ end or at 5’end, and whether a sequence spacer is needed between the two binding sites and what composition it should have (random?).
Thanks in advance
Laura
Is your biotinylated-oligo mimicking your small RNA of interest or it is used to fish your RNA of interest after specific binding?
Personnally, I'm fishing endogenous small RNA using such method. For this, I'm using a biotinylated antisens oligoribonucleotide containing a biotin in 3', separeted by a carbon chain as spacer (to avoid steric hyndrance). Such oligo were provided by IDT. They offer different spacer that can be used.
In addition, piece of RNA was made by 2'O-methylated ribonucleotides which confer a better stability.
Thank you Gabrielle for the suggestions!
My biotinylated-oligo is mimicking my RNA of interest. More precisely, I have to verify the ability of two RNA binding proteins to bind both wt and mutant forms of under study RNA. What the difference in adding biotin at 3' or 5' end?
In my opinion, the orientation doesn't matter as soon as your choice is not interfering biologically with your system. To explain, in my case my bait was carrying the biotin in 3' because I wanted to reduce the steric hindrance with the 3' of my prey. So, I made a choice knowing it would be the best orientation.
So I guess that if you don't know if the orientation might influence or not, maybe try both separately. It might be an option. In any case, I would highly recommend a spacer between the oligo and the biotin.
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