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  • synthase br Conclusions Peptide nanotubes were used to encap

    2018-11-13


    Conclusions Peptide nanotubes were used to encapsulate HRP and synthase also to facilitate direct synthase transfer between HRP redox sites and an electrode. To verify the effect of PNTs on electron transfer, a PNT/HRP system was tested using an amperometric method with hydrogen peroxide, and the result was compared with the case where HRP was immobilized without PNTs, and HQ was used as electron transfer mediator. These two experiments were compared to the case with both PNT and HQ and without any of them. The results are summarized as follows:
    Author contributions
    Conflicts of interest
    Introduction The nuclease S1 from Aspergillus oryzae has the ability to degrade single-stranded oligonucleotides composed of either deoxynucleotides or ribonucleotides [1,2]. In addition, it has been suggested to meanwhile possess activity that can recognize the loop portion of duplex and cleaves it, leaving a mono-ribonucleotide at the 5′ terminus of DNA strands [3,4]. Thus, it can be widely used in the DNA damage detection, nuclease protection assay, in removing single-stranded tails from DNA molecules and opening hairpin loops of double-stranded cDNA [5–8]. Until now, the active site and key structural elements necessary for DNA hydrolysis have been recognized through structural analysis of S1, while biochemical evidences for the function of S1 are still much limited. In order to exploring the mechanism of enzymatic reaction catalyzed by S1, developing simple and efficient methods for monitoring reaction still remains to be done. Several traditional methods for S1 detection include gel electrophoresis and autoradiography [9–11]. Due to the shortcomings of being time-consuming, labor-intensive and of insufficient sensitivity, great efforts have been made to develop new methods with better performance, such as fluorescence resonance energy transfer based fluorescent methods, enzymatic amplification based label-free fluorometric assay [12,13], nanomaterials [14–16] or G-quadruplex based colorimetric methods [17]. Although these methods have achieved great advances toward the S1 assay, there still are some limitations in each method. For example, dual-labeling of DNA probes with fluorescent dyes was required in the above-mentioned fluorescent methods. Multi-step modifications or washings were commonly existed in nanomaterial based assay. In G-quadruplex based assays, DNA probes need specific design. These weaknesses make the above assays expensive, complicated and/or tedious in some extent. Recently, graphene oxide, a new generation of nanomaterial with unique physical–chemical properties, shows high potential for use in the biomedical assay. It has been confirmed that the quenching efficiency of this fluorescence quencher is higher than those of existed quenchers [18,19]. In addition, the short single-stranded DNA had weaker affinity to GO than long ssDNA [20,21]. Thus, these properties make it possible to prepare a novel bioassay platform for monitoring DNA cleavage reaction catalyzed by nuclease. However, this kind of optimal quencher can inhibit some enzymes\' activity through spatial hindrance effect [19,22]. We also found that graphene oxide can inhibit S1 activity under the environment of acid pH when it was applied as the quencher of fluorescence probe (data not shown). That\'s maybe the reason why there was no method for monitoring S1 assay in real time. Interestingly, we found that S1 can efficiently digest graphene oxide quenched fluorescence probe in the neutral buffer containing Mg, which is consistent with previous report [23]. In addition, we sifted gentamycin sulfate as a strong stimulator of S1. These results provide the solid foundation for developing a simple and sensitive responsive sensor of S1 assay.
    Experimental
    Results and discussion
    Conclusion
    Acknowledgments This work was partially supported by the Natural Science Foundation of Hunan Province (h14JJ2049), the Natural Science Foundation of China (31201074) and the Science and Technological Program for Dongguan\'s Higher Education, Science and Research, and Health Care Institutions (2011108102026).