Fig. 2: Inhibition by EN inhibitors determined with a fluorescent oligonucleotide activity assay. | Nature Communications

Fig. 2: Inhibition by EN inhibitors determined with a fluorescent oligonucleotide activity assay.

From: Identification and characterization of small molecule inhibitors of the LINE-1 retrotransposon endonuclease

Fig. 2

a Schematic of the fluorescent oligonucleotide assay. Left: sequence of hairpin oligonucleotide containing 5’ 6-FAM fluorescein fluorophore (F) and 3’ DABCYL quencher (Q). Middle: arrowhead indicates location of nick by EN at the semi-specific target site sequence 5’-TTTT*A-3’. The melting temperature of the green sequence is lower than the reaction temperature, whereas the melting temperature of the full hairpin is higher than the reaction temperature. Right: the fluorescently-tagged nicked sequence (8 nucleotides) is released from the quencher sequence (35 nucleotides), allowing for fluorescence to occur as a real-time readout of EN activity. b Representative assay results for each EN inhibitor. Activity was determined as the initial rate of reaction under multiple turnover conditions. Graphs show the percent of no inhibitor control as a function of indicated inhibitor concentration. Average IC50 values ± s.d. were calculated using the four parameter [inhibitor] vs. response non-linear fit in GraphPad Prism for each of the 3 technical replicates (shown as circles, diamonds, and triangles along with corresponding curve fits) in order to obtain s.d. values. No inhibitor control and full inhibition by 50 mM EDTA were included in fit calculations to guide definition of top and bottom of curve fits. Average IC50 values calculated across at least 3 independent experiments can be found in Table 1. Source data are provided as a Source Data file.

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