Fast and simple purification of chemically modified hammerhead ribozymes using a lipophilic capture tag.
A new type of 5'-lipophilic capture tag is described, enabling the facile reverse phase HPLC purification of chemically modified hammerhead ribozymes (oligozymes) whilst still carrying the 2'-O-tert.-butyldimethylsilyl protection of the essential riboses. In its most convenient form, the capture tag consists of a simple diol, such as hexan-1,6-diol, which at one end is attached via a silyl residue to a highly lipophilic entity such as tocopherol (vitamin E) or cholesterol, and the other end is functionalized as a phosphoramidite. This lipophilic capture tag is added as the last residue in the solid-phase synthesis of chemically modified hammerhead ribozymes. Cleavage from the support and release of all protecting groups except for the silyl groups is achieved with ethanolamine/ethanol. The crude product is then loaded directly on to a reverse phase HPLC column. Separation of failure peaks from full length product is achieved easily using a short run time. The retarded product peak is collected, lyophilized, desilylated in the normal way and then desalted. This method removes the lipophilic capture tag yet leaves behind the hexanediol entity which helps protect the compound against degradation by 5'-exonucleases. The purity of the product as judged by analytical anion-exchange HPLC and capillary gel electrophoresis is generally better than 95% full-length, and yields of 2-4 mg from a 1 micromol scale synthesis are routine. In addition, the method can be readily scaled up, an important feature for the development of such chemically modified ribozymes as potential therapeutics.
Sproat
B S
BS
Innovir GmbH, Olenhuser Landstrasse 20b, D-37124 Rosdorf, Germany. brian.sproat@embl-heidelberg.de
Rupp
T
T
Menhardt
N
N
Keane
D
D
Beijer
B
B
D002784:Cholesterol; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000302:isolation & purification*; D013997:Time Factors; D014810:Vitamin E
D009841:Oligonucleotides; D016337:RNA, Catalytic; D014810:Vitamin E; D002784:Cholesterol