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

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.

One-Step Ligation on RNA Amplification for the Detection of Point Mutations

“…As the DP length is decreased, the specificity increases because the rate of ligation on mismatched RNA templates decreases at a faster rate than the rate of ligation on the target RNA template. Previously, 25 the use of [g-thio]-triphosphate as a substrate for T4 DNA ligase in DNA array profiling found a delayed rate of ligation reactions. We show that CPs with pre-adenylated 1-Thio-AMP groups improve the specificity of ligation reactions on RNA when mismatches are present.…”

Quantitative Approach for Small Molecules Using Laser Desorption/Ionization Mass Spectrometry

“…Surfaces are stabilized on the surface layer, and a minimal amount of matrix is required for measurement. 12) and inhibitors of the anthrax lethal factor. 13) The process may maximize the utility of LDI-MS, and its usefulness and future potential have been confirmed.…”

Discovery of SIRT3 Inhibitors Using SAMDI Mass Spectrometry

“…The SAMs can be customized to use a variety of immobilization chemistries, including maleimide and azide (or alkyne) for covalent capture, and biotin for streptavidin-biotin capture. [8][9][10] Monolayers specifically bind and capture the intended analytes, allowing for isolation of the analyte from 384 samples in parallel-by using the standard microtiter plates-and we are now transitioning to 1536-well plates. Furthermore, the intrinsic compatibility of SAMDI with essentially any enzyme activity (provided the activity results in a change in the molecular weight of the substrate) permits the screening of posttranslational modifications using peptide substrates.…”