Nucleic Acids Research Advance Access published online on October 23, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp827
Methods Online |
Profiling the selectivity of DNA ligases in an array format with mass spectrometry
Department of Chemistry, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637, USA
*To whom correspondence should be addressed. Tel: +1 773 702 1651; Fax: +1 773 702 1677; Email: mmrksich{at}uchicago.edu
Received May 2, 2009. Revised August 20, 2009. Accepted September 18, 2009.
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.
Present address: J. Kim, Department of Chemistry, Kyung Hee University, Seoul, South Korea.