Chapter 12 Using Fluorophore-Labeled Oligonucleotides to Measure Affinities of Protein–DNA Interactions

Abstract

Changes in fluorescence emission intensity and anisotropy can reflect changes in the environment and molecular motion of a fluorophore. Researchers can capitalize on these characteristics to assess the affinity and specificity of DNA-binding proteins using fluorophore-labeled oligonucleotides. While there are many advantages to measuring binding using fluorescent oligonucleotides, there are also some distinct disadvantages. Here we describe some of the relevant issues for the novice, illustrating key points using data collected with the F plasmid relaxase domain and a variety of labeled oligonucleotides. Topics include selection of a fluorophore, experimental design using a fluorometer equipped with an automatic titrating unit, and analysis of direct binding and competition assays.

Burkholderia xenovorans RcoM Bx -1, a Transcriptional Regulator System for Sensing Low and Persistent Levels of Carbon Monoxide

“…Strains of the greatest utility are available at the Addgene repository (www.addgene.org/). As described previously (22), B. xenovorans LB400 bears two RcoM paralogues, encoded by rcoM 1 and rcoM 2 , and these genes are transcribed divergently from the adjacent coxM 1 and coxM 2 genes, as illustrated for rcoM 1 and coxM 1 (see Fig. 1).…”

“…The designated "f" motif occurs adjacent to the putative 70 RNAP Ϫ35 binding sequence ( Fig. 1), and as demonstrated below, the "d ϩ e ϩ f" region plus the RNAP-binding site constitute a functional promoter where RcoM Bx -1 binding activates the expression of coxM 1 . This activity was monitored in vivo by creating coxM 1 =::lacZ transcriptional fusions (see supplemental methods and Fig.…”

“…Fluorescence polarization assays of RcoM Bx -1 binding to Texas Red (TR)-labeled DNA targets designated according to the "a" to "f" conventions (Table 1) were employed to assess binding under conditions where the protein form [heme Fe(III), or Fe(II) or Fe(II)-CO] was readily controlled, in order to unambiguously demonstrate CO-dependent binding. Unfortunately, while preliminary tests under various assay conditions with 0.1 to 8 M purified RcoM Bx -1 protein demonstrated CO-dependent binding, they failed to attain a limiting upper asymptote, which precluded the determination of a dissociation constant (1,28). This may reflect the propensity of LytTR domain proteins to aggregate (13,25,31,47) and/or the fact that a triple-binding-site probe was not tested.…”

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

“…1) and examined their interaction with FL-dsDNA 15 using the direct DNA binding assay (Fig. 5A) (26). Macroscopic binding constants, K N , were determined by fitting the titration curves using a one-site binding model (Equation 2) and are summarized in Table 4.…”

“…6D). The K int for T 25 in the absence of any nucleotides was determined to be 2.8 (Ϯ0.4) ϫ Effect of Ionic Strength on DNA Binding-To further define the DNA-binding properties of TraI, we examined the effect of ionic strength on the affinity of TraI for ssDNA and dsDNA using the direct DNA binding assay (26). Titration of 50 nM FL-T 25 was performed in buffer C supplemented with increasing concentrations of NaCl: 50, 75, 100, and 125 mM.…”