title: Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes.
abstract: Sequence-specific recognition of nucleic acids by proteins is required for nearly every aspect of gene expression. Quantitative binding experiments are a useful tool to measure the ability of a protein to distinguish between multiple sequences. Here, we describe the use of fluorophore-labeled oligonucleotide probes to quantitatively monitor protein/nucleic acid interactions. We review two complementary experimental methods, fluorescence polarization and fluorescence electrophoretic mobility shift assays, that enable the quantitative measurement of binding affinity. We also present two strategies for post-synthetic end-labeling of DNA or RNA oligonucleotides with fluorescent dyes. The approaches discussed here are efficient and sensitive, providing a safe and accessible alternative to the more commonly used radio-isotopic methods.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Pagano
authors.forename: Carina C
authors.initials: CC
authors.lastname: Clingman
authors.forename: Sean P
authors.initials: SP
authors.lastname: Ryder
mesh_terms: D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D009696:Nucleic Acids / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D011506:Proteins