Evaluation of coupling of cobalamin to antisense oligonucleotides by thin-layer and reversed-phase liquid...

We have evaluated by chromatography two strategies of oligonucleotide binding to vitamin B12 (cobalamin). The first one was based on a covalent linkage of aminooligonucleotide to carboxycobalamin in presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). Carboxycobalamin and EDC-cobalamin were eluted with a retention time of 16.5 and 21.6 min, respectively, in RP-HPLC, while aminooligonucleotide and oligonucleotide-cobalamin were coeluted at 19.4 and 19.8 min. In the second strategy, avidin was coupled to both biotinylated oligonucleotide and vitamin B12. Aminocobalamin and biotinylated cobalamin had respective retention times of 13 and 15.7 min in RP-HPLC and respective Rf values of 0.3 and 0.8 in thin-layer chromatography. Incubation of avidin with biotinylated cobalamin produced, in Superose 12 gel permeation, a peak with a retention time of 28 min, which corresponded to avidin-biotinylated cobalamin as it disappeared with an excess of either biotin or biotinylated oligonucleotide. In conclusion, we have prepared and purified by RP-HPLC and gel permeation chromatography an oligonucleotide-avidin-cobalamin complex which will be used as a vector complex of antisense oligonucleotides.