title: Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay.
abstract: We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1°C for (dT)5 to a maximum of 9°C with oligomers ⩾10 nucleotides, with an apparent Kd of <1μM for (dT)20. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9°C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner.
authors.affiliation: Department of Basic Medical Sciences, The University of Arizona College of Medicine-Phoenix, Phoenix, AZ 85004-2157, USA.
authors.forename: Kanchi Ravi
authors.initials: KR
authors.lastname: Rupesh
authors.affiliation: Department of Basic Medical Sciences, The University of Arizona College of Medicine-Phoenix, Phoenix, AZ 85004-2157, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Smith
authors.affiliation: Department of Basic Medical Sciences, The University of Arizona College of Medicine-Phoenix, Phoenix, AZ 85004-2157, USA. Electronic address: boehmer@email.arizona.edu.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Boehmer
mesh_terms: D004268:DNA-Binding Proteins / Q000737:chemistry*; D018259:Herpesvirus 1, Human / Q000737:chemistry*; D008024:Ligands; D055550:Protein Stability / Q000187:drug effects; D044366:Transition Temperature / Q000187:drug effects*
chemical_list: D004268:DNA-Binding Proteins; D008024:Ligands
keywords: Herpes simplex virus-1; ICP8; SYPRO Orange; Single-strand DNA binding protein; Thermal shift assay