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Assay of endonuclease activity plays an important role in biologic and medicinal study.Herein,a simple and facile strategy was developed for detecting endonuclease activity.In this strategy,poly(AT-TA)double-stranded DNA containing restriction cutting site for EcoRI endonuclease was rationally designed and acted as the substrate of EcoRI and the template for copper nanoclusters(CuNCs).