Attachment of hybridizable oligonucleotides to a silica support and its application for selective extraction of unmodified and antisense oligonucleotides from serum samples

摘要

The main aim of the present study was the synthesis of an oligonucleotide-based material with high chemical stability, repeatability and specificity to complementary oligonucleotides. The oligonucleotides were attached to a silica gel surface modified with amino acids during one-step synthesis. The amount of the oligonucleotides immobilized on the support surface had an impact on adsorption effectiveness, due to steric interference. The adsorption capacity corresponds to 4.7 mu g of complementary oligonucleotide per 1 mg of material, which reflects 50% of immobilized oligonucleotides. The presented results contain comprehensive studies on hybridization and release of fully complementary, partially complementary, non-complementary and antisense oligonucleotides from the newly synthesized adsorbent. The salt concentration and time period were the most influential parameters in the case of adsorption, while high temperature and low salt content were indispensable for effective desorption. Selectivity studies revealed that the adsorption percentage increases with the decreasing number of base mismatches. Consequently, the desorption of low complementarity oligonucleotides was always greater in comparison with the fully complementary sequence. Furthermore, it was shown that oligonucleotide-based materials may be successfully used for the extraction of antisense oligonucleotides and their metabolites from serum samples with recoveries ranging between 65 and 73%.