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

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 hig h 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 i mmobilization efficiencies were achieved, and at 2.7 M potassium phosp hate, 85-90% of the DNA initially present in the reaction mixture was immobilized. Reactions were examined for double stranded DNA, one stra nd of which was modified with a 5'-mercaptohexyl spacer arm, and for e ach of the strands comprising the duplex. For double stranded immobili zations, about 85% of the non-modified strand (the d22 strand) was rel eased from the support under melting conditions, suggesting that d22 e xhibited low reactivity when organized as the duplex. For immobilizati ons of single stranded DNA, mild acid hydrolysis of the products was u sed to provide information concerning the mode of attachment. For reac tions of the d22 strand alone, only about 60% each of guanine and aden ine were recovered from the immobilized oligonucleotide following mild acid hydrolysis. This suggests that when d22 is immobilized as the si ngle strand, significant attachment occurs through the purine bases, i n contrast with the low reactivity exhibited by d22 in the duplex. Pur ified 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 gra dient.