RNA Hybridization to DNA Coupled with Cyanogen‐Bromide‐Activated Sephadex

A method for purifying RNA by hybridization to complementary DNA immobilized on Sephadex is described and is illustrated by the purification of polyoma mRNA. Denatured DNA can be immobilized on cyanogen‐bromide‐activated Sephadex whilst remaining freely available for hybridization. However, after activation with cyanogen bromide, followed by the coupling of DNA and deactivation with ethanolamine, Sephadex acts as an ion‐exchange resin. It is therefore necessary to elute RNA from DNA‐Sephadex by a combination of formamide buffers containing NaCl and elevated temperatures, conditions which dissociate DNA · RNA hybrids and also prevent the binding of RNA to the resin by ion exchange. Polyoma DNA, or restriction fragments thereof, were bound to Sephadex and used to purify viral mRNA. RNA purified from polyadenylated RNA isolated from the cytoplasm of mouse cells 28–30 h after infection with polyoma virus sedimented at values between 19 and 16 S in sucrose/formamide gradients and, in a wheat germ cell‐free translation system, directed the synthesis of three polypeptides; these were identified by gel mobility, immunoprecipitation and fingerprinting as the three structural proteins of polyoma virus. Viral mRNA could also be purified by hybridization directly from total cytoplasmic infected‐cell RNA without prior selection of polyadenylate‐containing RNA. The advantages and limitations of DNA attached to cyanogen‐bromide‐activated Sephadex as an affinity matrix for RNA purification are discussed.