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• A new method has been proposed for monitoring PNK activity in vitro , natural compound screening and intracellular imaging. • NS17 isolated from Kadsura heteroclita screened as PNK inhibitor, which effect confirmed in vitro and in cell level. • NS17 can scavenge intracellular ROS, inhibit inflammatory cells proliferation and down-regulate COX-2 expression. Polynucleotide kinase (PNK) is critical for nucleic acid metabolism, as its overexpression can increase ROS production and activate the inflammation pathway, leading to chronic inflammation. In this study, we proposed a new fluorescence method for monitoring PNK in vitro , natural compounds screening and intracellular imaging. Quantitative detection depicted that the limit of detection (LOD) of PNK is 0.0021 U/mL in the linear range of 0.01∼10 U/mL. Methodological evaluation showed that both of the selectivity and the inhibitory effect of ADP and EDTA on PNK activity with satisfactory results. Fluorescence imaging of inflammatory cells demonstrated the reliability of platform for capturing PNK change in situ regulated by inhibitor of natural compound NS17 isolated from Kadsura heterolita. Mechanism study showed that the inhibitor NS17 can reduce inflammation by scavenging intracellular ROS, killing inflammatory cells and down-regulating COX-2 expression, the processes of which are related with PNK activity. In summary, above results suggest that this new platform could monitor PNK activity in situ and screen inflammation inhibitors targeted on PNK. [ABSTRACT FROM AUTHOR]

• The DNAzyme-rGO coupled fluorescence assay achieved ultra-sensitive detection of T4PNK. • This method shows great potential in inhibitor analysis, enzyme kinetic studies, and natural drug screening of T4PNK. • This is the first study to monitor T4 PNK activity in in living cells. • Activation of T4PNK by natural compounds was verified by imaging in HepG2 cells. Phosphorylation of DNA caused by T4 PNK is an important regulatory process that associated with many cellular events and diseases. Here, we proposed a DNAzyme-rGO coupled fluoresence method for T4 PNK detection. Under the optimal conditions, the approach exhibits high sensitivity with a detection limit of 0.001 U/mL, and there was a reliable linear relationship between fluorescence intensity and T4 PNK concentration in the range of T4 PNK from 0.001 U/mL ∼ 5 U/mL. Then, this method was used for kinetic analysis and effectors screening from natural compounds. Finally, the method was applied to monitor activity of T4 PNK in vitro and intracellular imaging of T4 PNK. In summary, this sensitive and specific sensing platform for T4 PNK assay can be hopefully used for clinical diagnosis, prognosis evaluation and targeted drug screening. [ABSTRACT FROM AUTHOR]

A label-free, ultra-sensitive and turn-on method for detecting RNase A has been developed using enhanced DNA-templated silver nanoclusters (DNA-AgNCs) as the fluorescence probe. In this system, an RNA strand, which can perfectly hybridize with DNA template of nanocluster synthesis, was applied to lock the fluorescent signal of DNA-AgNCs by forming an RNA/DNA duplex. Meanwhile, the hybridized RNA/DNA duplex was used as the substrate of RNase A. The fluorescence signal of AgNCs was restored due to the degradation of RNA by RNase A. From the fluorescence signal change of this system caused by RNase A, it was found that the fluorescence signal showed a positive linear relation with RNase A concentration in the range from 0.2 pg/μL to 10 pg/μL with a detection limit of 0.098 pg/μL. Except for potential inhibitor screening and the kinetic study of this enzyme, this strategy was further used for monitoring dynamic change of RNase A in living cells successfully. In summary, the simple and sensitive method for RNase A assay can be hopefully used for drug screening in vitro and in vivo. Image 1 • Synthetic DNA-enhanced silver nanoclusters display high and stable fluorescence optical properties. • This method demonstrates the high sensitivity and specificity of RNase A detection. • This strategy was successfully used to monitor dynamic changes of RNase A in living cells after screening for the potential inhibitor. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are regarded as important biomarkers for disease diagnostics and therapeutics due to their significant regulatory roles in physiologic and pathologic processes. Herein, a versatile nanoprobe based on reduced graphene oxide (rGO) and nucleic acid (DNA) probe was prepared for simultaneously visualize miR-451a and miR-214–3p in living cells. In vitro experiments demonstrated that the nanoprobe exhibits excellent selectivity and outstanding sensitivity as low as 1 nM towards miR-451a and miR-214–3p. Moreover, the detection signals of miRNAs have good linearity in their respective concentration ranges (miR-451a: 1–100 nM, Y 1 = 9.3062X 1 +114.85 (R2 = 0.9965). miR-214–3p: 1–200 nM, Y 2 = 1.4424X 2 +91.312 (R2 = 0.9961)). Finally, simultaneous dual-color imaging of miR-451a and miR-214–3p in human breast cancer cells (MDA-MB-231) was realized by exploiting the P 1 &P 2 @rGO nanoprobe. In summary, this simple and effective strategy provides a general sensing platform for highly sensitive detection and simultaneous imaging of dual miRNAs in living cells. Image 1 • Synthetic rGO displays prominent fluorescence quenching efficiency, excellent biocompatibility and strong anti-nuclease capability. • The nanosystem displays the ability of sensitive, selective, and facile detection of miRNAs. • The developed sensor is directly used for intracellular dual miRNA imaging，without the assistance of transfection reagent. [ABSTRACT FROM AUTHOR]