Abstract

Prokaryotic primase, a DNA-dependent RNA polymerase, is a target of interest for the development of novel antibiotics. A new assay was developed to evaluate the inhibition of primase activity while avoiding the limitations of existing assays that require the incorporation of radiolabeled nucleotides into the growing primer followed by electrophoretic separation and autoradiography or scintillation counting. These existing technologies are either time consuming or unable to give detailed information on the kinetics, size, and nature of the primers synthesized. To address these issues in a nonradioactive manner, a thermally denaturing high-performance liquid chromatography (HPLC) assay was developed that was able to (1) measure the two modes of primase activity (de novo and overlong primer synthesis), (2) quantitate de novo primer synthesis kinetics yielding a rate constant of 0.00251 s -1, and (3) determine that dNTPs inhibited primase activity with an IC50 of 9.5 μM. In addition, the differential elution properties of short DNA and RNA oligonucleotides on an alkylated nonporous polystyrene-divinylbenzene copolymer microsphere bead column were determined. The thermally denaturing HPLC assay provides rapid quantitative analysis of primase function and qualitative analysis of activity with regard to the nature of the primers synthesized.

Original languageEnglish (US)
Pages (from-to)330-336
Number of pages7
JournalAnalytical Biochemistry
Volume332
Issue number2
DOIs
StatePublished - Sep 15 2004

Fingerprint

DNA Primase
High performance liquid chromatography
High Pressure Liquid Chromatography
Assays
Scintillation Counting
Kinetics
Scintillation
DNA-Directed RNA Polymerases
Autoradiography
Microspheres
Oligonucleotides
Inhibitory Concentration 50
Rate constants
Nucleotides
RNA
Anti-Bacterial Agents
Technology
DNA
Chemical analysis

Keywords

  • DnaG primase
  • Enzyme Inhibitors
  • Enzymology
  • Escherichia coli
  • HPLC
  • Nucleic acid synthesis Inhibitors
  • Oligonucleotides

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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title = "Thermally denaturing high-performance liquid chromatography analysis of primase activity",
abstract = "Prokaryotic primase, a DNA-dependent RNA polymerase, is a target of interest for the development of novel antibiotics. A new assay was developed to evaluate the inhibition of primase activity while avoiding the limitations of existing assays that require the incorporation of radiolabeled nucleotides into the growing primer followed by electrophoretic separation and autoradiography or scintillation counting. These existing technologies are either time consuming or unable to give detailed information on the kinetics, size, and nature of the primers synthesized. To address these issues in a nonradioactive manner, a thermally denaturing high-performance liquid chromatography (HPLC) assay was developed that was able to (1) measure the two modes of primase activity (de novo and overlong primer synthesis), (2) quantitate de novo primer synthesis kinetics yielding a rate constant of 0.00251 s -1, and (3) determine that dNTPs inhibited primase activity with an IC50 of 9.5 μM. In addition, the differential elution properties of short DNA and RNA oligonucleotides on an alkylated nonporous polystyrene-divinylbenzene copolymer microsphere bead column were determined. The thermally denaturing HPLC assay provides rapid quantitative analysis of primase function and qualitative analysis of activity with regard to the nature of the primers synthesized.",
keywords = "DnaG primase, Enzyme Inhibitors, Enzymology, Escherichia coli, HPLC, Nucleic acid synthesis Inhibitors, Oligonucleotides",
author = "Koepsell, {Scott A} and Bastola, {Dhundy Raj} and Hinrichs, {Steven Heye} and Griep, {Mark A}",
year = "2004",
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language = "English (US)",
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N2 - Prokaryotic primase, a DNA-dependent RNA polymerase, is a target of interest for the development of novel antibiotics. A new assay was developed to evaluate the inhibition of primase activity while avoiding the limitations of existing assays that require the incorporation of radiolabeled nucleotides into the growing primer followed by electrophoretic separation and autoradiography or scintillation counting. These existing technologies are either time consuming or unable to give detailed information on the kinetics, size, and nature of the primers synthesized. To address these issues in a nonradioactive manner, a thermally denaturing high-performance liquid chromatography (HPLC) assay was developed that was able to (1) measure the two modes of primase activity (de novo and overlong primer synthesis), (2) quantitate de novo primer synthesis kinetics yielding a rate constant of 0.00251 s -1, and (3) determine that dNTPs inhibited primase activity with an IC50 of 9.5 μM. In addition, the differential elution properties of short DNA and RNA oligonucleotides on an alkylated nonporous polystyrene-divinylbenzene copolymer microsphere bead column were determined. The thermally denaturing HPLC assay provides rapid quantitative analysis of primase function and qualitative analysis of activity with regard to the nature of the primers synthesized.

AB - Prokaryotic primase, a DNA-dependent RNA polymerase, is a target of interest for the development of novel antibiotics. A new assay was developed to evaluate the inhibition of primase activity while avoiding the limitations of existing assays that require the incorporation of radiolabeled nucleotides into the growing primer followed by electrophoretic separation and autoradiography or scintillation counting. These existing technologies are either time consuming or unable to give detailed information on the kinetics, size, and nature of the primers synthesized. To address these issues in a nonradioactive manner, a thermally denaturing high-performance liquid chromatography (HPLC) assay was developed that was able to (1) measure the two modes of primase activity (de novo and overlong primer synthesis), (2) quantitate de novo primer synthesis kinetics yielding a rate constant of 0.00251 s -1, and (3) determine that dNTPs inhibited primase activity with an IC50 of 9.5 μM. In addition, the differential elution properties of short DNA and RNA oligonucleotides on an alkylated nonporous polystyrene-divinylbenzene copolymer microsphere bead column were determined. The thermally denaturing HPLC assay provides rapid quantitative analysis of primase function and qualitative analysis of activity with regard to the nature of the primers synthesized.

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