Assessment of DNase Activity by Ratiometric Fluorescence Resonance Energy Transfer

The present protocol describes a simple and sensitive ratiometric Förster resonance energy transfer (FRET) assay for the detection and quantitative assessment of DNase activity. Ratiometric FRET measurements make use of the ratio of donor and acceptor emission signals. The assay detects single-stranded DNA breaks using a staple-shaped, dual-tagged 38-mer FRET oligoprobe, employed as a real-time DNA cleavage sensor. The main application of the described approach is the quantitative assessment of the effects of reaction conditions, such as pH, temperature, and buffer composition, on DNase activity. Due to its ability to detect even minor and slow DNA cleavage, the assay is particularly well suited for investigating weak nucleolytic activity requiring extended periods of observation and for studying the effects of pH on DNase activity. These specific advantages of this ratiometric FRET protocol are illustrated by its application to the detection and analysis of the DNase activity of leukocyte elastase inhibitor (LEI).