Assays for the determination of the activity of DNA nucleases based on the fluorometric properties of the YOYO dye

•Two assays for monitoring DNA digestion by λ-exonuclease and EcoRV in real-time.•Assays rely on the different fluorescence intensities between ss- and ds-DNA–YOYO.•Sensitive determination of nuclease activity and quantification of reaction products.

Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA–protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA–YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect.

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