In the past few years

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 element 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 RITA sale 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.
Experimental
Results and discussions
Conclusion
Introduction The joining of DNA fragments using DNA ligase is an essential process in gene cloning. One of the important parameters for performing DNA ligation efficiently is the temperature [1]. In the case of DNA strands with cohesive ends, ligation is generally performed at 12–16°C since higher temperatures may reduce the ligation efficiency by melting annealed DNA ends [1], [2], [3]. The ligation of blunt-ended DNA is usually carried out at room temperature with a high concentration of T4 DNA ligase [1], [4]. The optimal temperature of commonly used DNA ligase is around 37°C and therefore the above ligation conditions are not optimal for the action of DNA ligase. Several attempts to increase the ligation efficiency by improving thermal conditions for the reaction have been reported. Lund, Duch and Pedersen [2] reported that a high enzyme activity and DNA annealing were balanced by constant temperature cycling, as a result of which the ligation efficiency was increased. Ranjan and Rajagopal [3] demonstrated that DNA fragments with 2-bp overhangs were ligated using T4 DNA ligase at room temperature with high efficiency when the DNA fragments were subjected to heating followed by flash freezing prior to the ligation reaction. It was recently reported that an enzyme immobilized on ferromagnetic particles was heated and activated under a radio frequency alternating magnetic field due to heat dissipation from the particles caused by magnetic hysteresis and eddy currents with no influence on the activity of free enzyme around the particles [5]. This suggests that DNA ligase can be activated utilizing the heat generation of ferromagnetic particles subjected to an ac magnetic field with little effect on the annealing of DNA ends. In this article, we carry out the ligation of DNA fragments with cohesive ends using T4 DNA ligase immobilized on ferromagnetic particles and show that the ligation efficiency is increased by applying an ac magnetic field. The basic concept of our method is illustrated in Fig. 1. DNA ligase is immobilized on the surface of ferromagnetic particles and a reaction solution containing the DNA ligase/ferromagnetic particle hybrids and DNA fragments to be ligated is set at low temperature suitable for the annealing of DNA ends. When a radio frequency alternating magnetic field is applied, the ferromagnetic particles generate heat caused by magnetic hysteresis and eddy currents [6], [7], [8], [9], [10], [11] and DNA ligase on the particles is selectively heated up and activated. If we optimize the experimental conditions, the activation of DNA ligase can be carried out almost without melting annealed DNA ends. The present method is so simple that it can easily be combined with other methods for efficient DNA ligation including the addition of molecular crowding agents such as polyethylene glycol [12], [13], [14] or hexamine cobalt chloride [15].