A DNAzyme-rGO coupled fluorescence assay for T4PNK activity in vitro and intracellular imaging

Journal Pre-proof A DNAzyme-rGO coupled fluorescence assay for T4PNK activity in vitro and intracellular imaging Ruxin Luo, Hongyan Zhou, Wenya Dang, Ying Long, Chunyi Tong, Qian Xie, Muhammad Daniyal, Bin Liu, Wei Wang

PII:

S0925-4005(20)30231-8

DOI:

https://doi.org/10.1016/j.snb.2020.127884

Reference:

SNB 127884

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

19 November 2019

Revised Date:

8 February 2020

Accepted Date:

13 February 2020

Please cite this article as: Luo R, Zhou H, Dang W, Long Y, Tong C, Xie Q, Daniyal M, Liu B, Wang W, A DNAzyme-rGO coupled fluorescence assay for T4PNK activity in vitro and intracellular imaging, Sensors and Actuators: B. Chemical (2020), doi: https://doi.org/10.1016/j.snb.2020.127884

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A DNAzyme-rGO coupled fluorescence assay for T4PNK activity in vitro and intracellular imaging Ruxin Luo1, Hongyan Zhou1, Wenya Dang1, Ying Long1, Chunyi Tong1, Qian Xie2, Muhammad Daniyal2, Bin Liu1*, Wei Wang2* 1

College of Biology, Hunan University, Changsha, 410082, P. R China

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TCM and Ethnomedicine Innovation & Development International Laboratory, Innovative Material Medical Research

Institute, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, 410208, P. R China

E-mail: [email protected] (B. Liu); [email protected] (W. Wang)

Research Highlights

Due to its bioassay high catalytic activity, simple synthesis, high thermal stability, design

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To whom correspondence should be addressed. Tel: +86-731-89720939; Fax: +86-731-89720939;

flexibility and reduced nonspecific adsorption, DNAzymes used in this article is an ideal signal

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amplification tool.

Reduced grapheme oxide (rGO) shows strong fluorescence quenching capacity

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The DNAzyme-rGO coupled fluorescence assay achieved ultra-sensitive detection of T4PNK.

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This method shows great potential in inhibitor analysis, enzyme kinetic studies, and natural drug screening of T4PNK.

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This is the first study to monitor T4 PNK activity in in living cells.

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Activation of T4PNK by natural compounds was verified by imaging in HepG2 cells.

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Abstract

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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 1

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.

Keywords: Fluorescence assay, T4 PNK, DNAzyme, rGO, Natural compounds screening, Intracellular imaging

1. Introduction T4 polynucleotide kinase (PNK), which can catalyze the transfer of γ-phosphate residue of ATP to the 5’hydroxyl group of oligonucleotides and nucleic acids, plays a pivotal role in the 5’-kinase family [1, 2], as the 5’-

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hydroxyl terminal phosphorylation of DNA is a vital regulatory process involving most typical cellular events, including DNA replication, recombination, and DNA damage repair [3-6]. Many evidences have clearly illustrated that abnormal activity of T4 PNK may impede DNA phosphorylation, which is the main cause of certain important human diseases such as Rothmund-Thomson syndrome, Werner syndrome and Bloom syndrome, et al [6, 7]. Given

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the significant role of T4 PNK in these pathological processes, it is imperative to develop novel methods so as to quickly and specifically obtain important information for diagnosis and diseases therapy. 32

P-labeling, autoradiography and polyacrylamide gel

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To date, several kinds of methods including radioactive

electrophoresis for the PNK activity detection in vitro have been developed [8, 9]. Nevertheless, these approaches

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suffered from several intrinsic shortcomings, including sophisticated instruments, cumbersome operating procedures, and a potential threaten to the human health. Recently, many novel analytical methods for T4PNK have been

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established, such as fluorescence detection [10-12], electrochemical methods [13], colorimetry assay, and chemiluminescence or bioluminescence methods [14]. Among them, fluorescence-based methods showed significant advantages of sensitivity, simplicity, low-cost and quantitative feasibility, which have drawn wide attention. For

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instance, Tang et al. established a fluorescence method for monitoring phosphorylation process by using MB-based “phosphorylation-ligation” coupled enzyme reaction [15], Jiao et al. described a non-labeling perylene probe for

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PNK activity assay [16], Zhou et al. developed a new strategy for T4 PNK by combining reduced graphene oxide quenched fluorescent probe with ligase reaction [10]. However, these fluorescence methods still existed the problem of unsatisfactory sensitivity. So, it is still necessary to propose alternative platforms, particularly with high sensitivity and convenience, for evaluating the activity of T4 PNK. DNAzyme is a class of DNA molecules with catalytic functions [17]. Like protein and RNA catalytic enzymes, DNAzymes can catalyze many types of biochemical reactions and are widely used in asymmetric catalysis [18], biosensors [19], DNA nanotechnology [20], and clinical diagnostics [21]. In order to improve the sensitivity of 2

biosensing system, signal amplification effect became the most important choice. Many researchers have realized the improvement of sensitivity of sensing events by using enzymes like endonuclease and exonuclease as biocatalysts to amplify detection signal. Compared with general protein enzymes, DNAzyme exhibited several advantages such as high catalytic activity, simple synthesis, high thermal stability, design flexibility and reduced nonspecific adsorption [22-24]. These obvious advantages make them ideal assistance tools for the assay of various biomolecules and metal ions an so on. With the continuous development of nanotechnology, different nanomaterials have been applied in various fields [25]. Moreover, graphene, which outlook is considered to be a revolutionary material, displays excellent optical, thermal conductivity properties, and has broad application prospects [17]. As the oxide form of

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electrical and

graphene, graphene oxide has been widely used in drug delivery due to its good biocompatibility, large specific surface area and easy surface modification [26]. In addition, using the ultra-high fluorescence quenching ability, acceptable biosafety and high stability in various solution, GO and its derivative of rGO have been reported for

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biosensing assay and molecule imaging [27]. In the proposed method, based on the signal amplification of DNAzyme

was constructed to achieve the detection of T4 PNK.

2.1 Materials and chemicals

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2. Experiment section

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and the differential quenching ability of rGO for fluorescent probes of different lengths, a novel sensitive biosensor

T4 polynucleotide kinase (No.2021A) and ATP (No.4041) were purchased from Takara Biotechnology Co. Ltd

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(Dalian, China). Lambda exonuclease (λ-exo) (No.M0262) was purchased from New England Biolabs Co. Ltd (Beijing, China). Sequences information of all oligonucleotides (Takara Biotechnology Co. Ltd) were shown in Table.S1. The natural compounds were isolated from Panax Japonicus C.A meye (LW2、LW4、LW5) and Swertia

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punicea Hemsl (SZ1、SZ2、SZ4、SZ6) by ourselves. The details structure and information of them were indicated in Table.S2. The reaction buffer was consisted of 50 mM Tris-HCl (pH 8.0) and 10 mM MgCl2.

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2.2 Fluorescence assay for T4PNK activity In the phosphorylation assay, samples with 100 μL volume were consisted of 100 nM HP, 1 mM ATP and T4 PNK with various concentration. After incubating at 37 °C for 50 min, the samples were denatured at 75 °C for 10 min, then cooled to 37 °C for 10 min to ensure enough phosphorylation of the substrate. To perform the λ-exo-mediate cleavage reaction, 5 μL NaCl (50 nM) and 4 μL λ-exo (0.02 U/μL) were added successively and incubated at 37 ℃ for 50 min. After producing DNAzyme, 1 μL FAM-labled probe (100 nM) were added into solution and incubated at 37 ℃ for 50 min. Then, the above mixtures were separately incubated with 3 μL rGO (final concentration of 3 μg/mL) 3

at 37 ℃ for 15 min. Finally, the samples (Ex/Em=450/521nm) were measured on the FL-2500 fluorescence spectrophotometer (Hitachi, Japan). 2.3 Specificity analysis 0.05 U/μL Uracil - DNA Glycocasylase (UDG), Apurinic/apyrimidinic endonuclease (APE1), T4 DNA Ligase, DNase1, Ribonuclease H (RNase H), and T4 PNK were adopted to explore the specificity of the designed T4 PNK assay. The reaction conditions and procedures were consistent with the aforementioned T4 PNK activity detection. 2.4 Kinetic study of T4 PNK For T4 PNK kinetics study, reactions were carried out with HP concentrations varying from 0.025 ~ 0.25 μM,

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incubated for 2 min at 37 ℃and then incubated for 10 min at 75 ℃. Other conditions are the same as described above. 2.5 Inhibition study of T4PNK

To study the inhibitory effect of inhibitor on T4 PNK activity, two well-known inhibitors of EDTA and ADP were selected to perform the inhibition analysis by using T4 PNK detection system. The concentration of the inhibitors

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was 0, 2, 4, 6, 8 and 10 mM for EDTA, 0, 0.5, 1, 1.5, 2 and 3 mM for ADP. The experiment procedures were the same as previously described.

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2.6 Natural compounds screening

To explore the effect of natural compounds on the T4 PNK activity, 5 μL T4 PNK(0.05 U/μL), 1 μL ATP(1mM)

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and 4 μL 7 kinds different natural compounds (20 μM ) were added and incubated at 37 ℃ for 10 min and then coincubated with HP. The remaining procedures were consistent with above mentioned for T4 PNK assay. To explore

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the effect of natural compounds on λ-exo, 5 μL NaCl (50 nM) and 4 μL different natural compounds (20 μM) were added to the solution as the control sample and incubated at 37 ℃ for 10 min. After that, phosphorylated HP (p-HP)

described.

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were added into the above solution and incubated at 37 ℃ for 50 min. The following steps were the same as previously

2.7 Molecular modeling

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Molecular docking was carried out by applying software of Molecular Operating Environment (MOE.2015.10.) to investigate the mechanism of interaction between natural compounds and T4 PNK. First, ChemBioDraw was applied to convert natural compounds into 3D structures to import MOEs, and a database of these compounds was then established. Next, to obtain a stable 3D structure, the 3D structure introduced in the .sdf format was protonated and the energy of the compound was minimized and saved as the .moe format. Then, creating a new database in the MOE, and importing the .moe format file into the .mdb format file. Meanwhile, the sequence of T4 PNK from the NCBI database (GenBank: AHY83916.1) were downloaded to establish a T4 PNK crystal structure model in .pdb format. 4

After importing the .pdb format into the MOE software, the excess ligand group were deleted and the dummy active binding site were predicted. Finally, the dummy active binding site was docked with the compound to obtain molecular docking results. Repeatedly, T4 PNK was docked with other compounds according to the above procedure. 2.8 T4 PNK assay in cell-free extracts The human hepatocarcinoma cells of HepG2 (cell Bank of the Xiangya Central Laboratory, Central South University) were seeded in 6-well cell culture plate for preparing cell extracts. Subsequently, the cells were devided into different group: (1) control group; (2) LW5 treatment group (final concentration of 10 μM, 20 μM); (3) SZ4 treatment group (final concentration of 10 μM, 20 μM).

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The preparation process of cell-free extract was as follows: tumor cells with above treatment were firstly treated with trypsin followed with the addition of cell lysate buffer. Then, the cells were lysed on ice bath for 35 min before with ultrasonic treatment at 55 W for 3 min on the homogenizer (Ningbo Scientz Biotechnology Co. Ltd.). Finally, the absorbance value at 562 nm was measured to perform quantitative analysis. For T4PNK assay, the diluted cell

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extract (1 mg/mL) was added to the reaction system and the detection procedures were the same as described above. 2.9 Cell imaging

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Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum and 1% antibiotics were used to culture HepG2 cells in a 12-well dish. SZ4 and LW5 were separately added to two wells and incubated for 24 h, the

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other cells without treatment as the control. HP, lambda exonuclease and DNAzyme were mixed with liposomes (liposomes were diluted with DMEM) respectively. Many reports proved that liposomes have lysosomal escape

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function[28, 29], thus liposomes can protect them from degradation by lysosomes. Then, 1% FBS was added to 200 μL and incubated at 25 °C for 20 min. After discarding the medium and washing with warm PBS for 3 times, the above mixture was separately added to the cells. Meanwhile, 200 μL 1% FBS was directly added into the control

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well. Then, all cells were incubated at 37 °C for 2.5 h. Subsequently, the mixture of probe1, rGO and DMEM was incubated at 25 °C in the dark for 30 min, and 200 μL of the mixture was added to each well.

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Adding above solution to the dish and incubating at 37 °C for 4 h, then discarding old medium and washing with PBS. Ultimately, the cells were treated with 180 μL of Hoechst 33342 (Yeasen Biotech Co. Ltd.) for 1 h. After washing with cold PBS, confocal microscopy images of living cells were acquired on the Olympus FluoView FV1200 laser-scanning microscope（Olympus, Japan).

3. Results and discussion 3.1 Principle for T4PNK detection The principle of the new method for T4 PNK assay is demonstrated as Fig.1. We designed a hairpin-shaped DNA 5

probe-HP as the substrate of T4 PNK. As a highly processive exonuclease, λ-exo can recognize 5’-phosphoryl termini and cleavage dsDNA from 5’o 3’ end, while exhibits ignorable cleavage activity to dsDNA with a 5’-hydroxyl end. Thus, HP probe with the hydroxyl group at 5’-terminus cannot act as the substrate of λ-exo [30]. However, the introduction of ATP and T4 PNK can cause phosphorylation of HP at the 5’ end and the corresponding product can be cleaved by λ-exo to yield active DNAzyme. Subsequently, in the presence of Mg2+, the cleavage of single-strand fluorescence probe by DNAzyme will produce short FAM-labeled fragments, which cannot be absorbed by rGO, thereby emitting fluorescence. Eventually, one DNAzyme molecule can trigger many cleavage cycles, causing the amplification of fluorescence signal. However, the lack of T4 PNK will keep the status of HP probe with hydroxyl

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group at 5’end. The unphosphorylated substrate cannot be digested by λ-exo and cannot produce active DNAzyme to cleave the FAM-labeled probe. The presence of rGO caused high efficient fluorescence quenching of the probe between them. Thus, T4 PNK activity monitoring can be conveniently realized in vitro and in vivo by utilizing anti

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nuclease ability of rGO.

Fig .1 Schematic illustration for T4PNK activity detection

3.2 Feasibility analysis First, the effect of T4 PNK presence on the fluorescence signal of whole system was investigated in order to confirm the strategy. The fluorescence spectra in Fig.2A indicated that the fluorescent signal of the FAM-labeled probe (probe1) with rGO was quenched about 87% due to the splendid quenching ability of rGO. After adding T4 6

PNK, the fluorescence intensity recovered about 53%. The intuitionistic histogram result in Fig.2B further directly indicated that the signal of the sample added with T4 PNK is closely to that of sample containing phosphorylated HP (p-HP) and the sample adding with DNAzyme. This result indicated that the successful phosphorylation of HP 5’terminus by T4 PNK specifically recognized by λ-exo and yielded active DNAzyme to induce fluorescence recovery

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by digesting its specific substrate. Thus, these results clearly indicated the feasibility of the assay for T4 PNK activity.

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Fig.2 (A) The waveform of fluorescence emission spectra. (B) The histogram of fluorescence emission spectra. [p-HP] = [HP] = [Probe1] = [DNAzyme] = 100 nM. [T4 PNK] = 0.05 U/μL, [ATP] = 1 mM, [λ-exo] = 0.02 U/μL and [rGO] = 3 μg/mL.

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3.3 Optimization of the detection conditions

Considering the need to obtain optimal reactive conditions for achieving sensitive detection of T4 PNK, we investigated some crucial factors that can affect the system’s sensing efficiency. Fig.3A indicated that the intensity

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gradually decreased with the increase of T4 PNK concentration. Moreover, the highest fluorescence signal was obtained at the presence of 3 μg/mL rGO. Therefore, the optimized concentration of rGO was determined as 3 μg/mL.

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Subsequently, by investigating the effect of λ-exo concentration on the fluorescence intensity, it was found that the increase of fluorescence intensity almost reached equilibrium at the presence of 0.02 U/μL λ-exo (Fig.3B). Thus, we

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picked this concentration as s. The phosphorylation time and the amount of ATP were also optimized as both of them were pivotal parameters for the phosphorylation process of T4 PNK. Fig.3C and Fig.3D respectively indicated that the fluorescence intensity reached equilibrium quickly after phosphorylation for 50 min and the addition of 1 mM ATP. Consequently, phosphorylation for 50 min and 1 mM ATP were taken as the optimal conditions for the assay. In addition, Fig.3E indicated that the relative fluorescence intensity increased as the pH value of the buffer increased, and it reached the peak at a pH value of 8.0. The continuing increase of pH value more than 8.0 decreased the fluorescence intensity conversely. As a result, pH 8.0 was applied on the following experiments. 7

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Fig.3 The effects of different factors on the T4 PNK assay. (A) The responses of T4 PNK sensing system to the rGO concentration. (B) The responses of T4 PNK sensing system to different concentrations of λ-exo. (C) The responses of T4 PNK sensing system to the T4

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PNK phosphorylation time. (D) The responses of T4 PNK sensing system to ATP concentrations. (E) The responses of T4 PNK sensing system to different pH. [HP] = [Probe1] = 100 nM, [T4 PNK] = 0.05 U/μL.

3.4 Sensitivity of the sensing method

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After obtaining optimal reactive conditions, we then discussed the influence of T4 PNK concentration on the fluorescence intensity of the new method. Fig.4A revealed that as the concentrations of T4 PNK changed from 0.001

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to 50 U/mL (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20 and 50 U/mL), the overall fluorescence intensity progressively increased. Meanwhile, Fig.4B depicted a reliable linear relationship between the fluorescence signal

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and T4 PNK concentration over a range from 0.001 U/mL ~ 5 U/mL with a linear regression equation of y= 94.561x + 711.45, and the correlation coefficient was 0.9871. In the inset of Fig. 4B, there was a plot for the quantification of T4 PNK activity, and the limit of detection was estimated to be 0.001 U/mL, which was superior to most of reported methods (Table.S3). Therefore, the newly developed method achieved ultra-sensitive detection of T4 PNK activity.

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Fig.4 (A) The fluorescence emission spectra of the detecting system at various concentration of T4 PNK. (B) The fitting curve of T4 PNK activity assay. The insert curve in (B) shows the linear relationship between (F-F0) and lg ([T4 PNK]), F and F0 represent the fluorescence intensity of the detection system at different concentrations of T4 PNK and T4 PNK, respectively. Samples contained 100 nM HP, 1 mM ATP, 0.02 U/μL λ-exo, 100 nM probe1 and 3 μg/mL rGO.

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3.5 Selectivity of the strategy

To investigate the specificity of the T4 PNK detecting method, we studied the effect of some enzymes, including

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UDG, APE1, T4 DNA ligase, DNase1 and RNase H. Fig.5 clearly illustrated that the system produced dramatic increase of fluorescence intensity only after adding of T4 PNK, whereas negligible change was observed for the

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presence of other enzymes. This result indicated that the above enzymes have negligible effect on the detecting

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system, demonstrating a high selectivity of the sensing system toward T4 PNK.

Fig.5 [T4 PNK], [UDG], [APE1], [T4 DNA Ligase], [DNase1] and [RNase H] are 0.05 U/μL, respectively. [HP] = [Probe1] = 100 nM. [T4 PNK] = 0.05 U/μL, [ATP] = 1 mM, [λ-exo] = 0.02 U/μL and [rGO] = 3 μg/mL.

3.6 Kinetic analysis The application of the assay for T4 PNK kinetic study was then performed. Different concentrations of substrate 9

HP (0.0025~0.05) and 0.05 U/μL T4 PNK were added in the sample at 37 ℃. The results in Fig.6A showed a positive correlation between the initial velocity and substrate concentration. By Linewaver-Burk analysis, Km was calculated to be 38.08 nM. These reliable results confirmed that the method can fulfill the requirement of kinetic analysis of T4

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PNK.

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Fig.6 (A) Enzymatic reaction rate responses to various concentration of HP. (B) The Lineweaver-Burk plot of T4 PNK. [probe1] = 100 nM, [T4 PNK] = 0.05 U/μL, [ATP] = 1 mM, [λ-exo] = 0.02 U/μL and [rGO] = 3 μg/mL.

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3.7 Inhibitor analysis of T4PNK

Since abnormal T4 PNK activity often resulted in the development of severe human diseases [6, 7], T4 PNK

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inhibitors screening is meaningful for drug development and clinical therapeutics. First, we chose EDTA and ADP, which were reported as inhibitors, as models for inhibiting ability evaluation. It has been reported that EDTA could

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inhibit T4 PNK activity by weakening its binding ability to substrate [31], while ADP inhibits the phosphorylation by affecting the transfer efficiency of phosphate groups [32]. Fig.7 demonstrated the fluorescence decrease within the increase of EDTA and ADP concentrations. The IC50 of EDTA and ADP were calculated as 4.3 mM and 2.36 mM,

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respectively, similar to previous report data [33]. This result effectively suggested that the new detection system can

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be used for reliably screening T4 PNK inhibitors.

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Fig. 7 Inhibition effects of (A) EDTA, (B) ADP on T4 PNK activity. [HP] = [Probe1] = 100 nM. [T4 PNK] = 0.05 U/μL, [ATP] = 1 mM, [λ-exo] = 0.02 U/μL and [rGO] = 3 μg/mL.

3.8 Natural compounds screening

In order to broaden the application of this method, it was used for T4 PNK targeted natural compounds screening.

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Herein, we selected several natural compounds with anti-inflammatory effects as model drugs. Fig. 8A showed that compounds under low dose (20 μM) simultaneously indicated the regulation function on the T4 PNK activity and

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lambda exonuclease. After eliminating the interference of lambda exonuclease, 6 drugs (LW2, LW4, LW5, SZ1, SZ2, SZ4) still showed evident active effect on the T4 PNK activity. Among these drugs, SZ4 showed the strongest effect

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(Fig.8B). Then, we used SZ4 as the target to study the relationship between the concentration and the activation effect of T4 PNK and obtained positive relation between SZ4 concentration and fluorescence intensity. This result implied

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that SZ4 has a concentration-dependent enhancement effect on the T4 PNK activity in vitro (Fig.8C). Then, we further carried out Molecular docking studies by using MOE software tried to disclose natural compounds’ active mechanism. Molecular docking results of SZ4 was shown as Fig.8D&E and others were shown

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in Table.S4. In accordance with previous studies, the active sites of T4 PNK were identified as Lys 15, Ser16 and Arg126 [1]. All drugs directly bind with the active amino acid, and the natural compound except SZ6 had a docking

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score of more than 30 with T4 PNK. Thus, it can be concluded that the activation of T4 PNK was achieved by the interaction between compounds with the active center of this enzyme. Meanwhile, these results further confirmed that Molecular docking is an efficient method for screening T4 PNK-related drugs in medicinal chemistry field.

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Fig. 8 (A) Effects of natural compounds on T4 PNK and λ-exo. (B) Effects of natural compounds on the entire detection system. (C) Concentration dependence of natural compound SZ4. (D) The ligand- interaction diagram of natural compound SZ4 with T4 PNK. (E)

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The binding pose of natural compound SZ4 with the 5’-kinase domain of T4 PNK. [Compounds] = 20 μM. [p-HP] = [HP] = [Probe1] =

3.9 Detection of T4PNK in complicated biosamples

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100 nM. [T4 PNK] = 0.05 U/μL, [ATP] = 1 mM, [λ-exo] = 0.02 U/μL and [rGO] = 3 μg/mL.

Subsequently, we used this method to detect T4 PNK in biosample to explore its practical application. We explored

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the effect of the two compounds on the T4 PNK activity of human hepatoccarcinoma cell line HepG2. Fig.9 indicated that both of them upregulated T4 PNK activity of tumor cells in a concentration-dependent manner. Since the

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abnormal PNK activity of is relevant to many diseases including tumor, we suspect that the two drugs can play their protecting function from liver cell damage by upregulating PNK activity. Nevertheless, the concreate mechanism

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needs further study.

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Fig. 9 T4 PNK detection in cell extracts. [Cell extracts] = 0.01 mg/mL. [HP] = [Probe1] = 100 nM. [T4 PNK] = 0.05 U/μL, [ATP] = 1 mM, [λ-exo] = 0.02 U/μL and [rGO] =3 μg/mL.

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3.10 Cell imaging of T4PNK in vitro

Eventually, the novel approach was used to monitor the activity of T4 PNK through intracellular imaging. In this

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section, HepG2 was still used as the model for enzyme imaging in vivo. First, by investigating the effect of time on the fluorescence imaging, it was found that after incubating probe1 and rGO for 4 hours (Fig.S1), the fluorescent

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signal was the strongest, so 4 h was chosen as the optimal time point. Meanwhile, the cells incubating with different components showed differential green fluorescence in vivo and the fluorescence is mainly localized in cytoplasm rather than in the nucleus, which were consistent with our expectation. Moreover, tumor cells incubating with

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substrate (HP) and λ-exo or DNAzyme showed strong green fluorescence (Fig.10A&B). In addition, SZ4 and LW5 of T4 PNK activators were used to treat HepG2 cells. As we expected, the fluorescence intensity was evidently

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improved in the compounds treated cells comparing with that of control (Fig.10C&D). However, those cells simply incubating with probe and rGO only showed weak fluorescent signals (referred as background signal). (Fig.10E). This result indicated that SZ4 and LW5 can upregulate T4 PNK activity in HepG2 cells, which is in accord with natural drug screening experiments in vitro. Therefore, these results demonstrated that the new method provide a useful alternative for screen T4 PNK targeted drugs from cell level.

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Fig. 10 Confocal microscopy images of HepG2 cells incubating with different natural compounds. [HP] = [DNAzyme] = 300 nM,

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[SZ4] = [LW5] = 20 μM, [probe1] = 400 nM, [λ-exo] = 0.02 U/μL and [rGO] = 6 μg/mL.

4. Conclusion

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In summary, the developed T4 PNK detecting method was demonstrated to be very facile and sensitive, which possessed quite a few exceptional characteristics including lower limit of detection, wide liner rang and good selectivity. In addition, the inhibitor screening, enzyme kinetic study, and drug screening of T4 PNK were successfully.

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Moreover, this method was applied for T4 PNK activity monitoring in living cells. According to our point, this method is expected to provide a potential platform for T4 PNK sensing in clinical diagnosis and therapy.

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Credit Author Statement

Ruxin Luo performed this article, analyzed the data and wrote this paper. Hongyan Zhou designed and

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guided the experiment. Wenya Dang helped performed the experiment and evaluated the data. Ying Long made recommendations on experimental data analysis. Dr. Chunyi Tong provided constructive comments on the experiment and program design. Qian Xie isolated and provided natural compounds. Muhammad Daniyal helped modified the language of the manuscript. Dr. Wang Wei supported the experiment and made valuable comments on the article. Dr. Bin Liu proposed the idea, supervised the experiment and modified the manuscript.

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Conflict of interest The authors declare that they have no conflict of interest.

Declaration of interests

The authors declare that they have no known competing financial interests or personal

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relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

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This work was partially supported by the Natural Science Foundation of China (81673579 and 31672457).

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Author Biographies Ruxin Luo received a bachelor's degree from college of Life Sciences, Guangxi Normal University. She is commencing her master study in College of Biology, Hunan University from 2018 under the guidance of Prof. Bin Liu. Her current research interests include biosensing assay. Hongyan Zhou received her bachelor's degree from college of Medicial, Hunan Normal University. She is currently pursuing her PhD degree under the guidance of Prof. Chunyi Tong from the College of Biology, Hunan University. Her main research interests are focused on biosensing assay and

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nanotheranostics. Wenya Dang earned her bachelor's degree from School of Life Sciences, Shanxi Datong University. She is commencing her master study in College of Biology, Hunan University from 2017 under the

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guidance of Prof. Bin Liu. Her research focus on the molecular diagnosis and biosensing assay.

Ying Long is currently a post-doctor and research associate in College of Biology, Hunan University.

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She received her PhD degree in Pathology and Pathophysiology from Xiangya Hospital, Central South University in 2018. Her research interests include biochemistry and molecular biology, proteomics

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and bioinformatics and nanotheranostics.

Prof. Dr. Chunyi Tong is currently an associate professor in Hunan University in PR China. He

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received his B.S. and PhD degree in Analytical Chemistry from Hunan University in 2003 and 2008, respectively. From 2016–2017, he was a visiting scholar at the University of Pennsylvania. His major

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research interests are focused on biosensors, nanotheranostics and nanoantibacterial.

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Qian Xie received her master degree from the School of Pharmacy at Hunan University of Chinese Medicine in 2014 and now working on her PhD degree at the same university under the guidance of Dr. Wei Wang. She had worked as a visting scholar in National Center for Natural Products Research, University of Mississippi for half a year. She focus on the isolation and discovery of bioactive natural products from Chinese medicine or other ethnic medicine, as well as nanotheanostics. Muhammad Daniyal is a PhD fellow at Hunan University of Chinese Medicine working on natural compounds screening, anti-cancer therapy, mechanism studies of natural 19

compounds, and development of nano drug delivery system.

Prof. Dr. Bin Liu is currently an associate professor in Hunan University in PR China. He received his PhD degree in Analytical Chemistry from Hunan University at 2007. From 2007 to 2009, he was a post-doctor and research associate in Internal Medicine School, Health Sciences Center, Texas Tech University. His major research interests focus on the biosensors, nanotheranostics and nanoantibacterial.

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Prof. Dr. Wei Wang is Furong Distinguished Professor of Hunan Province, China and director of TCM and Ethomedicine Innovation & Development International Laboratory of Hunan University of Chinese Medicine. He received his Ph.D. degree from Peking University in 2006. He had worked in National Center for Natural Products Research, University of Mississippi from 2007 to 2012. He and

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his group focus on the isolation and discovery of novel bioactive natural products from Chinese

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medicine or other ethnic medicine, design of new analytical chemistry methodology as well.

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