Goodisman J, Dabrowiak J C
Department of Chemistry, Syracuse University, NY 13210.
J Biomol Struct Dyn. 1985 Feb;2(5):967-79. doi: 10.1080/07391102.1985.10507613.
In the footprinting experiment, an end-radiolabeled DNA restriction fragment is subjected to digest by an endonuclease in the presence and absence of a ligand which alters the endonuclease cleavage rate at sites of ligand-DNA contact. The location of these sites, and the strength of the ligand binding, are then deduced from the measured concentrations of the different oligonucleotides produced by the digest. We analyze the experiment in terms of coupled kinetic equations which take into account the cutting rates of endonuclease for sites with ligand present and absent, and the rates of binding and dissociation of the ligand to a site. As long as the ligand concentration remains essentially constant (which occurs, for example, if digest is terminated early enough to assure that all fragments result from single cuts by the endonuclease), the oligonucleotide concentrations reflect only the ligand binding equilibrium constant (ratio of rate constants) and the cutting rates in the presence and absence of ligand. We also show how the measured oligonucleotide concentrations (from, e.g. an autoradiogram) can be used to deduce the ligand equilibrium binding constants for the various sites on the polymer.
在足迹实验中，一个末端放射性标记的DNA限制片段在有和没有配体存在的情况下接受核酸内切酶的消化，该配体会改变核酸内切酶在配体与DNA接触位点的切割速率。然后，根据消化产生的不同寡核苷酸的测量浓度推断这些位点的位置以及配体结合的强度。我们根据耦合动力学方程来分析该实验，这些方程考虑了核酸内切酶对有配体和无配体位点的切割速率，以及配体与位点的结合和解离速率。只要配体浓度基本保持恒定（例如，如果消化足够早地终止以确保所有片段都来自核酸内切酶的单次切割，就会出现这种情况），寡核苷酸浓度仅反映配体结合平衡常数（速率常数之比）以及有配体和无配体时的切割速率。我们还展示了如何使用测量的寡核苷酸浓度（例如来自放射自显影片）来推断聚合物上各个位点的配体平衡结合常数。
