Profiling the selectivity of DNA ligases in an array format with mass spectrometry

Joohoon Kim, Milan Mrksich*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

This article describes a method for the global profiling of the substrate specificities of DNA ligases and illustrates examples using the Taq and T4 DNA ligases. The method combines oligonucleotide arrays, which offer the benefits of high throughput and multiplexed assays, with mass spectrometry to permit label-free assays of ligase activity. Arrays were prepared by immobilizing ternary biotin-tagged DNA substrates to a self-assembled monolayer presenting a layer of streptavidin protein. The array represented complexes having all possible matched and mismatched base pairs at the 3' side of the nick site and also included a number of deletions and insertions at this site. The arrays were treated with ligases and adenosine triphosphate or analogs of the nucleotide triphosphate and then analyzed by matrix-assisted laser desorption-ionization mass spectrometry to determine the yields for both adenylation of the 5'-probe strand and joining of the two probe strands. The resulting activity profiles reveal the basis for specificity of the ligases and also point to strategies that use ATP analogs to improve specificity. This work introduces a method that can be applied to profile a broad range of enzymes that operate on nucleic acid substrates.

Original languageEnglish (US)
Article numbergkp827
Pages (from-to)e2-e2
JournalNucleic acids research
Volume38
Issue number1
DOIs
StatePublished - Oct 23 2009

ASJC Scopus subject areas

  • Genetics

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