A new affinity purification procedure for DNA-binding proteins using bromoacetyl agarose.

Characterization of proteins binding to the promoters of eukaryotic genes has proved essential for understanding the transcriptional regulation of viral and cellular genes. Because of the low abundance of these proteins in the cell, conventional purification of these molecules has been laborious. In contrast, sequencespecific DNA affinity chromatography has greatly faciliated rapid isolation and characterization of DNA-binding proteins. A number of procedures for coupling specific DNA sequences to resins have been reported to be effective (1-5). However, all have restrictions on the form of DNA which can be attached to the matrix. In case of avidin or streptavidin columns, biotin must be coupled to the DNA (1,2). Cyanogen bromide activated coupling requires single-stranded overhangs (3). A more recent technique by Larson and Verdine allows efficient coupling of blunt-ended double-stranded ODNs, but requires the use of complex modified nucleosides during DNA synthesis (4). In most laboratories, a variety of double-stranded probes are available from gel shift analyses. It would be highly desirable to have a method which allows direct use of these double-stranded DNA probes for preparation of protein affinity purification columns. Recently, fast and efficient chemical autoligation in aqueous media between short oligomers terminated by thiophosphoryl and bromoacetamido groups has been reported (6,7). Here we describe the use of this chemistry for the simple, rapid and efficient attachment of any double-stranded DNA to a solid support The DNA loading procedure consists of the following two steps: (i) thiophosphorylation of a double-stranded DNA with •y-thio-ATP to generate a 5'-thiophosphoryl group and (ii) coupling of the thiophosphorylated DNA to bromoacetyl agarose resulting in efficient attachment of DNA to the resin. The derivatized resin was then used for the DNA affinity protein isolation. The scheme is shown as follows: