Abstract
A theory for deriving drug-DNA site binding constants from footprinting data is presented. Plots of oligonucleotide concentration, as a function of drug concentration, for various cutting positions on DNA are required. It is assumed that the rate of cleavage at each nucleotide position is proportional to the concentration of enzyme at that nucleotide and to the probability that the nucleotide is not blocked by drug. The probability of a nucleotide position not being blocked is calculated by assuming a conventional binding equilibrium for each binding site with exclusions for overlapping sites. The theory has been used to evaluate individual site binding constants for the antiviral agent netropsin toward a 139 base pair restriction fragment of pBR-322 DNA. Drug binding constants, evaluated from footprinting data in the presence of calf thymus DNA and poly(dGdC) as carrier and in the absence of carrier DNA, were determined by obtaining the best fit between calculated and experimental footprinting data. Although the strong sites on the fragment were all of the type (T.A)4, the value of the binding constant was strongly sequence dependent. Sites containing the dinucleotide sequence 5'-TA-3' were found to have significantly lower binding constants than those without this sequence, suggesting that an adenine-adenine clash produces a DNA structural alteration in the minor groove which discourages netropsin binding to DNA. The errors, scope, and limitations associated with the method are presented and discussed.
Highlights
The extension of quantitative footprinting analysis to small ligand-DNA studies appears straightforward, certain characteristics of these ligands complicate the extraction of thermodynamic data
If either the ligand-DNA or the probe-DNA equilibrium were disturbed during the experiment, the concentrations of oligonucleotides produced in the footprinting digests would not reflect the true binding isotherms for the various site loading events taking place in the system
In this paper we present the first quantitative analysis of DNase I footprinting titrations involving netropsin and show how values may be derived for site-specific drug binding constants on a DNA restriction fragment
Summary
The extension of quantitative footprinting analysis to small ligand-DNA studies appears straightforward, certain characteristics of these ligands complicate the extraction of thermodynamic data. If either the ligand-DNA or the probe-DNA equilibrium were disturbed during the experiment, the concentrations of oligonucleotides produced in the footprinting digests would not reflect the true binding isotherms for the various site loading events taking place in the system. It has been shown (Ward et al, 1987, 1988) that binding of drugs to DNA causes an increase in DNase I cleavage away from the site of binding. In this paper we present the first quantitative analysis of DNase I footprinting titrations involving netropsin and show how values may be derived for site-specific drug binding constants on a DNA restriction fragment. In addition to defining the scope and limitations of the method, we present new evidence that netropsin can discriminate between high-affinity sites of the type (A-T), in a sequence-dependent fashion
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