pyrophosphate and 5-carboxytetramethylrhodamine-succinimidyl-ester

Carbon nanotubes (CNTs) can efficiently quench the fluorescence of the adsorbed fluorophores and nonconvalently interact with soft single-stranded DNA (ssDNA). Upon disruption of CNTs-fluorescent oligonucleotides hybrid by nuclease S1, fluorescence turn-on was observed. Using this strategy, a platform based on fluorescence signal for monitoring the activity of nuclease with advantages of high sensitivity and commonality was established, and a linear relationship between initial cleavage reaction rate and nuclease S1 concentration is found in the range of 0.6-8.0 U mL(-1) with a detection limit of 0.08 U mL(-1). Furthermore, by taking pyrophosphate as an example, we use the assay to evaluate the prohibition effect on nuclease, and the extent of fluorescence recovery decreased linearly with increasing the concentration of pyrophosphate in the range of 0.2-1.4 mM, implying that the cleavage reaction by nuclease S1 was prohibited, and therefore this fluorescence assay can also be conveniently utilized for inhibitor screening of nuclease.

Topics: Diphosphates; DNA Cleavage; DNA, Single-Stranded; Enzyme Inhibitors; Fluorescent Dyes; Kinetics; Nanotubes, Carbon; Oligonucleotides; Rhodamines; Single-Strand Specific DNA and RNA Endonucleases; Spectrometry, Fluorescence