Guy M, Olszewski A, Monhoven N, Namour F, Guéant J L, Plénat F
Laboratory of Cellular and Molecular Pathology in Nutrition, EP CNRS 0616, Faculty of Medicine, Vandoeuvre-lès-Nancy, France.
J Chromatogr B Biomed Sci Appl. 1998 Feb 27;706(1):149-56. doi: 10.1016/s0378-4347(97)00534-3.
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.
我们已通过色谱法评估了寡核苷酸与维生素B12（钴胺素）结合的两种策略。第一种策略是在1-乙基-3-(3-二甲基氨基丙基)-碳二亚胺盐酸盐（EDC）存在的情况下，将氨基寡核苷酸与羧基钴胺素进行共价连接。在反相高效液相色谱（RP-HPLC）中，羧基钴胺素和EDC-钴胺素的保留时间分别为16.5分钟和21.6分钟，而氨基寡核苷酸和寡核苷酸-钴胺素在19.4分钟和19.8分钟时共洗脱。在第二种策略中，抗生物素蛋白与生物素化的寡核苷酸和维生素B12都进行了偶联。氨基钴胺素和生物素化的钴胺素在RP-HPLC中的保留时间分别为13分钟和15.7分钟，在薄层色谱中的Rf值分别为0.3和0.8。在Superose 12凝胶渗透柱上，抗生物素蛋白与生物素化的钴胺素孵育产生了一个保留时间为28分钟的峰，该峰对应于抗生物素蛋白-生物素化的钴胺素，因为它会在过量的生物素或生物素化的寡核苷酸存在下消失。总之，我们已通过RP-HPLC和凝胶渗透色谱法制备并纯化了一种寡核苷酸-抗生物素蛋白-钴胺素复合物，该复合物将用作反义寡核苷酸的载体复合物。
