New strategies for assessing the sequence selective binding of small molecules to DNA

Abstract

We have developed two different approaches towards overcoming limitations of footprinting.

REPSA: (Restriction Enzyme Protection Selection and Amplification) is a novel technique used to determine the binding sites of DNA binding ligands. This technique uses type IIs restriction enzymes (which cut several bases away from their recognition sites) to selectively cleave unbound DNA, while DNA fragments to which the ligand is bound are protected from digestion. The reaction is performed with oligonucleotides containing recognition sites for these enzymes which flank a central region of random bases around their cutting site. The uncut products of the reaction are amplified by PCR, and are subjected to further rounds of drug binding and enzyme cleavage, enriching for oligonucleotides containing the preferred ligand binding sites. After several rounds of selection the fragments are cloned and sequenced, revealing the preferred binding sites. We have successfully employed this technique for confirming the binding sites of TANDEM (TpA) and distamycin (AT), though it has been less successful for determining the binding sites of echinomycin (CpG).

MULTISITE: We have prepared a DNA footprinting substrate which contains all 136 possible tetranucleotide sequences, and have used DNase I footprinting to examine the binding of some ligands with known recognition properties. These ligands include actinomycin, which is known to bind to GpC; echinomycin (CpG), TANDEM (TpA), mithramycin (GpC, GpG) and the AT-selective minor groove binding ligands distamycin and Hoechst 33258. These ligands show large differences in their binding to tetranucleotides which contain the preferred dinucleotide steps. For example ACGT and ACGG are much better binding sites for echinomycin than GCGC, CCGG and GCGG.

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