Development of non-electrophoretic assay method for DNA ligases and its application to screening of chemical inhibitors of DNA ligase I

Daekyu Sun, Rheanna Urrabaz

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

A new rapid assay method for DNA ligases has been developed, which allows direct quantification of enzyme activity without using the traditional polyacrylamide gel electrophoretic technique. In this method, the 5′-biotinylated nicked duplex was used as a substrate for the ligase reaction, in which the 5′-end of the first oligonucleotide (19-mer) on the nicked strand is biotinylated and the second oligonucleotide (20-mer) on the same strand is labeled with radioactive 32P at the 5′-end. After ligation of the biotinylated 19-mer oligonucleotide into the second oligonucleotide with the reaction of DNA ligases, the biotinylated 19-mer oligonucleotide is converted into the radioactive 39-mer oligonucleotide. The ligase reaction products were heat-denatured to release both ligated and unligated biotinylated oligonucleotides. The biotinylated oligonucleotides were then captured on a streptavidin-coated microtiter plate and counted. The results obtained using this method correlated very well with those from the standard assay method using electrophoresis. Using this assay method, we were able to screen a chemical library and identify new DNA ligase inhibitors structurally related to resorcinol, which has growth inhibitory effects on the human breast cancer cell, MCF-7. The method described here is anticipated to be very useful for screening DNA ligase inhibitors from chemical libraries.

Original languageEnglish (US)
Pages (from-to)49-59
Number of pages11
JournalJournal of Biochemical and Biophysical Methods
Volume59
Issue number1
DOIs
StatePublished - Apr 30 2004
Externally publishedYes

Fingerprint

DNA Ligases
Oligonucleotides
Assays
Screening
Small Molecule Libraries
Ligases
Streptavidin
DNA Ligase ATP
MCF-7 Cells
Enzyme activity
Electrophoresis
Reaction products
Ligation
Hot Temperature
Cells
Breast Neoplasms

Keywords

  • DNA ligase assay
  • DNA ligase inhibitor
  • DNA ligases

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

Cite this

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abstract = "A new rapid assay method for DNA ligases has been developed, which allows direct quantification of enzyme activity without using the traditional polyacrylamide gel electrophoretic technique. In this method, the 5′-biotinylated nicked duplex was used as a substrate for the ligase reaction, in which the 5′-end of the first oligonucleotide (19-mer) on the nicked strand is biotinylated and the second oligonucleotide (20-mer) on the same strand is labeled with radioactive 32P at the 5′-end. After ligation of the biotinylated 19-mer oligonucleotide into the second oligonucleotide with the reaction of DNA ligases, the biotinylated 19-mer oligonucleotide is converted into the radioactive 39-mer oligonucleotide. The ligase reaction products were heat-denatured to release both ligated and unligated biotinylated oligonucleotides. The biotinylated oligonucleotides were then captured on a streptavidin-coated microtiter plate and counted. The results obtained using this method correlated very well with those from the standard assay method using electrophoresis. Using this assay method, we were able to screen a chemical library and identify new DNA ligase inhibitors structurally related to resorcinol, which has growth inhibitory effects on the human breast cancer cell, MCF-7. The method described here is anticipated to be very useful for screening DNA ligase inhibitors from chemical libraries.",
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