Hydroxyl radical modification for probing ribose moieties

Hydroxyl radical modification for probing ribose moieties 

Place 10 pmol renatured RNA or complex in 25 pi modification buffer on ice. 

Extract once with phenol, once with phenol/chloroform (1 1), and once with chloroform. 

Precipitate with EtOH and wash with 80% EtOH. 

The insensitivity to different RNA structures combined with the small size of the hydroxyl radical makes it a powerful probe for footprinting proteins on RNA. In particular protein interactions with minor groove, since both Cl and C4 are located in the minor groove of helices. The stability of protein-RNA complexes should be tested after modification (see Section 4.2) since hydroxyl radicals also react with proteins. 

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