Does anyone have experience on RNA biotin modification using T4 RNA ligase? Is it possible to add biotin to the RNA-protein complex's RNA part directly if the RNA have a free 3' end? If yes, how to prevent the RNA's self ligation? If not, is there any other method I can use to modify RNA part of a RNA-Protein complex?
Hello Qiang,
Yes using T4 RNA ligase you can end level the RNA. We use the thermoscientific RNA 3' End Biotinylation Kit. The details of the protocol you can find here http://www.piercenet.com/instructions/2162226.pdf
This protocol works fine and we have also checked the biotinylaiton by doing northern analysis with streptavidin HRP conjugate.
All the best
We published a method to purify RNP-complexes using biotinylated RNA at their 3' end.
Pongidi-Fix et al., 2013 Biochemical Journal,
http://www.ncbi.nlm.nih.gov/pubmed/23140180
The protocol will vary substantially depending if you want to label the RNA in a native RNP complex, of if you can first label the RNA, then assemble the complex. The ideal way would be to first label the RNA, then assemble your RNP. Unfortunately, if you want to do the former, label a native complex, Franck Martin's nice paper and the Thermoscientific Kit might not help you much. In fact, I'm not sure there is a straightforward way to label a native RNP by biotinylation of the RNA component. But I'm sure with some ingenuity it could be done, I would imagine.
Perhaps just add a short 3' biotinylated oligo with a 5' phosphate and T4 RNA ligase to your RNP in a ligation conducive buffer at 4C overnight. You should get some ligation. Then run over a column of some sort to remove the small unligated oligo (the thermo kit might be great because the biotinylated cytidine would be very small and easy to remove) and then use streptavidin to capture your complex. If its a complex mixture and you are trying to label your specific complex, you will need to try a sort of splint or overhang hairpin to get specific ligation. This is all hypothetical though, and people simply don't try and do this sort of experiment, in my experience.
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