A Simple, High-Resolution Method for Establishing DNA Binding Affinity and Sequence Selectivity

Abstract

Full details of the development of a simple, nondestructive, and high-throughput method for establishing DNA binding affinity and sequence selectivity are described. The method is based on the loss of fluorescence derived from the displacement of ethidium bromide or thiazole orange from the DNA of interest or, in selected instances, the change in intrinsic fluorescence of a DNA binding agent itself and is applicable for assessing relative or absolute DNA binding affinities. Enlisting a library of hairpin deoxyoligonucleotides containing all five base pair (512 hairpins) or four base pair (136 hairpins) sequences displayed in a 96-well format, a compound's rank order binding to all possible sequences is generated, resulting in a high-resolution definition of its sequence selectivity using this fluorescent intercalator displacement (FID) assay. As such, the technique complements the use of footprinting or affinity cleavage for the establishment of DNA binding selectivity and provides the information at a higher resolution. The merged bar graphs generated by this rank order binding provide a qualitative way to compare, or profile, DNA binding affinity and selectivity. The 96-well format assay (512 hairpins) can be conducted at a minimal cost (presently ca. $100 for hairpin deoxyoligonucleotides/assay with ethiduim bromide or less with thiazole orange), with a rapid readout using a fluorescent plate reader (15 min), and is adaptable to automation (Tecan Genesis Workstation 100 robotic system). Its use in generating a profile of DNA binding selectivity for several agents including distamycin A, netropsin, DAPI, Hoechst 33258, and berenil is described. Techniques for establishing binding constants from quantitative titrations are compared, and recommendations are made for use of a Scatchard or curve fitting analysis of the titration binding curves as a reliable means to quantitate the binding affinity.

Study on the binding interaction between perfluoroalkyl acids and DNA

“…The curves were linearized by Scatchard analysis (Boger et al 2001). The dye was mixed with DNA at a stoichiometric ratio and the PFAAs were determined from the fluorescence intensity values.…”

Acid–base properties of functionalised tripodal polyamines and their interaction with nucleotides and nucleic acids

“…18 This is the reason for the relatively small decrease in basicity observed in every one of the six first protonations of all three ligands. These stepwise protonation constants are in all cases lower than those reported for precursor L 1 , which can be attributed to the electron withdrawing character of the 25 pyridine and imidazole rings 17,18,19 The next three basicity constants of L 5 -L 7 can be ascribed to the protonation steps of the pyridine and imidazole rings attached to the arms. Acidbase behavior of ligand L 4 has been previously reported.…”

“…Formation of these protonated species can be explained by means of the basicity of the ligands. The ligand with imidazole groups is more basic, so the protonation of the aromatic nitrogen is produced 25 at a higher pH. Ligand L 4 has their nitrogen atoms in pyridine moieties protonated at more acidic pH.…”

“…As an 5 alternative method for estimation of affinity, at least as a comparison of ability of studied molecules to compete for binding with classical intercalators already bound to dspolynucleotides, 25 we have performed ethidium bromide (EB) displacement assays ( Figure S5, ESI † ). 10 The obtained IC 50 = 1.2 -0.15 suggest that affinities of L 4 -L 7 toward ct-DNA and poly A-poly U are comparable to the affinity of EB.…”

“…Much attention has focused on the design of organic DNA-binding agents as well as on the improvement of DNA detection methods in real time with high sensitivity. 2,3 Despite the large 25 number of cellular roles that RNA plays in biological processes, this macromolecule has been considered only recently an attractive target for therapeutic intervention. 4 RNA is essential for replication, 5 transcription 6 and regulation processes, 7 protein function 8 and catalysis.…”