In the past few years DNA templated fluorescent metal nanopa

In the past few years, DNA-templated fluorescent metal nanoparticles have been developed as ideal alternatives to organic dyes due to their facile synthetic process, outstanding optical properties, ultrafine size, and fine biological compatibility. Since the first successful demonstration of DNA-templated silver nanoclusters (AgNCs) by Dickson\'s group in 2004 (Petty, 2004), DNA-templated upconversion nanoparticles (UCNPs), quantum dots (QDs), and noble-metal nanoparticles (e.g., Au, Ag and Cu nanoclusters) have been intensely investigated in the past few years (Zhou et al., 2014, Ma et al., 2009, Liu, 2014, Liu et al., 2012, Shah et al., 2012, Shah et al., 2016, Zhu et al., 2016, Xu et al., 2016). Among them, DNA-templated CuNPs exhibit distinctive properties: (1) the synthetic speed of DNA-CuNPs was fast, which could be completed within several minutes; (2) the request of template DNA concentration for CuNPs is very low (at nM); (3) the MegaStokes shifting (～260nm) of CuNPs could reduce the background signal of complex samples and provide an opportunity for biological diagnostic techniques; (4) because copper is a essential trace RO4987655 for all organisms, the applications of CuNPs should be safer than other metal nanoparticles. As development of dsDNA-templated CuNPs by Mokhir and co-workers (Rotaru et al., 2010), we have systematically investigated the effect of sequence type and sequence composition on the formation of DNA-CuNPs. The result showed that single stranded poly T could also template CuNPs with excellent fluorescence, while dsDNA-templated CuNPs was poly (AT-TA)-dependence formation (Qing et al., 2013, Qing et al., 2014a). After then, poly T-templated CuNPs and dsDNA-templated CuNPs were employed as a powerful signal transducing unit for biochemical analysis, respectively (Qing et al., 2014b, Mao et al., 2015, Hu et al., 2016, Yang et al., 2017, Chi et al., 2017). In view of the distinct features and great potential in many fields of fluorescent CuNPs, we herein report a label-free and sensitive strategy for evaluating enzymes involved in DNA repair pathways via dumbbell-shaped DNA templated fluorescent CuNPs. In this strategy, a dumbbell-shaped DNA probe (DP) that consists of a poly (AT-TA) double-helical stem, two ploy T single stranded loops, and a nick point in the middle of stem, was smartly designed to highly-efficient template for fluorescent CuNPs’ formation. Then, this highly-efficient fluorescent CuNPs was applied to monitor ligase and PNK activity on the base of target-depended locking of dumbbell-shaped DNA. In the presence of targets, dumbbell-shaped DNA was locked and could resist the digestion of exonucleases, which could template bright CuNPs. However, in the absent of T4 PNK or T4 ligase, nicked DP underwent hydrolysis of exonucleases to mononucleotide and failed to act as a template for synthesizing fluorescent CuNPs. As a consequence, the fluorescence changes of CuNPs could be used to evaluate T4 PNK and T4 ligase activity. This label-free method is not only meaningful for further research on the ligase-mediated DNA repair progress but also valuable to nucleic acid phosphorylation related research.