Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay

Kanchi Ravi Rupesh, Aaron Smith, Paul E Boehmer

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 <1 μM 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.

Original languageEnglish (US)
Pages (from-to)604-608
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume454
Issue number4
DOIs
StatePublished - Nov 28 2015

Fingerprint

DNA-Binding Proteins
Human Herpesvirus 1
Freezing
Melting point
Assays
Stabilization
Hot Temperature
Oligomers
Ligands
Temperature
DNA
Nucleotides
Fluorescence
Ciprofloxacin
Viruses
Oligonucleotides
Carrier Proteins
Throughput
Therapeutics

Keywords

  • Herpes simplex virus-1
  • ICP8
  • Single-strand DNA binding protein
  • SYPRO Orange
  • Thermal shift assay

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay. / Rupesh, Kanchi Ravi; Smith, Aaron; Boehmer, Paul E.

In: Biochemical and Biophysical Research Communications, Vol. 454, No. 4, 28.11.2015, p. 604-608.

Research output: Contribution to journalArticle

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