SALT-INDUCED IMMOBILIZATIONS OF DNA OLIGONUCLEOTIDES ON AN EPOXIDE-ACTIVATED HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC AFFINITY SUPPORT

摘要

Synthetic oligonucleotides, possessing a recognition sequence for the transcription factor NF-KB, were immobilized onto an epoxide-activated hydroxyethylmethacrylate HPLC affinity support in the presence of high concentrations of potassium phosphate. The extent of immobilization increased with salt concentration in a manner analogous to that which has been reported for salt-induced immobilizations of proteins. High immobilization efficiencies were achieved, and at 2.7 M potassium phosphate, 85-90% of the DNA initially present in the reaction mixture was immobilized. Reactions were examined for double stranded DNA, one strand of which was modified with a 5'-mercaptohexyl spacer arm, and for each of the strands comprising the duplex. For double stranded immobilizations, about 85% of the non-modified strand (the d22 strand) was released from the support under melting conditions, suggesting that d22 exhibited low reactivity when organized as the duplex. For immobilizations of single stranded DNA, mild acid hydrolysis of the products was used to provide information concerning the mode of attachment. For reactions of the d22 strand alone, only about 60% each of guanine and adenine were recovered from the immobilized oligonucleotide following mild acid hydrolysis. This suggests that when d22 is immobilized as the single strand, significant attachment occurs through the purine bases, in contrast with the low reactivity exhibited by d22 in the duplex. Purified p50 protein, the DNA binding element of NF-kappa B, and nuclear extracts from phorbol ester-stimulated HeLa cells were injected onto a column packed with the double stranded product. In both cases p50 was retained on the column and was recovered upon elution with a salt gradient.