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Unexpectedly, CDs treated plasmid DNA with nicked open-circular configuration maintains the genetic activity in cell-free system which involves coupled gene transcription and translation. The gene expression of CDs treated plasmid DNA is enhanced compared with untreated plasmid DNA, which is analogous to the regulation effect of topoisomerase I on gene expression. Our work demonstrates the enzymatic enantioselectivity of chiral carbon dots, and the topoisomerase I mimicking activity will enable important and promising applications. © 2020 WILEY-VCH