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Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x

One-Strand Oligonucleotide Probe for Fluorescent Label-free “Turn-on” Detection of T4 Polynucleotide Kinase Activity and its Inhibition Fu Zhou,a Guangfeng Wang*a,b , Dongmin Shi,a Yue Sun,a Liang Sha, a Yuwei Qiua and Xiaojun Zhang*a,b

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Thioflavin T (ThT), as one of the most exciting fluorogenic molecules, boosting the “molecular-rotor” ability to induce DNA sequences containing guanine repeats to fold into G-quadruplex structures, has been demonstrated to sense the change by its remarkable fluorescence enhancement. In this work, taking T4 polynucleotide kinase (PNK) as a model, ThT/Gquadruplex based platform and λexonuclease (λexo) cleavage reaction were combined to design a label-free “turn-on” strategy for fast, simple and accurate detection of T4 PNK activity and its inhibition. In the presence of T4 PNK, the designed thioflavin T based molecular beacon (TMB) DNA probe could be phosphorylated and then digested by the cleavage of λexo, releasing the G-quartets, which was arrested by ThT adopting G-quadruplex with an obvious fluorescence generation for the “turn-on” detection of T4 PNK. In comparison to the traditional methods, the as-proposed TMB probe is convenient without any sophisticated labeling and separation processes but with high analytical performance. It exhibits a satisfying detection result for the activity of T4 PNK with a low detection limit of 0.001U mL-1, which is not only meaningful for further research on the disease-related biochemical process but also valuable to the molecular-target therapies.

1.Introduction Since the Human Genome Project (HGP) has comprehensively unveiled the full sequence of human genomes, DNA (Deoxyribonucleic acid) is no longer confined to its essential role as the human genetic information carrier due to its systematic and comprehensive researches in science and technology.1 Great advances of DNA-related studies have been witnessed in both nanotechnology,2 material science,3 molecular computing4 and bioanalysis5. Molecular Beacons (MBs),as a novel class of fluogenic DNA based detection agents, were firstly reported in 1996 by Tyagi.6 They have manifested excellent performance for applications in numerous in vitro hybridization assays7-9 and a variety of non-nucleic acid targets such as small molecules and proteins10,11 utilizing aptamer-substrate recognition. Classical MBs are dual-labeled with fluorophore and quencher groups at the 5’ and 3’ ends respectively and can self-assemble into a stem-loop structure, such as hairpin and other secondary structures.6,7,12,13,14 Before binding to the target, the stem of MBs holds the two moieties in proximity to each other, causing the fluorescence to be a.

Anhui Key Laboratory of Chem-biosensing, College of Chemistry and Materials Science, Center for Nanoscience and Nanotechology, Anhui Normal University, Wuhu, 241000, P.R. China. E-mail: [email protected]; wangyuz@ mail.ahnu.edu.cn b. State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha,410082, P.R. China. † See DOI: 10.1039/x0xx00000x

quenched by fluorescence resonance energy transfer (FRET). Then a subsequent conformational change is induced when MBs-target complexes form, which opens the stem separating the fluorophore and quencher moieties and reactivating the fluorescence. Admittedly, although conventional MBs have been widely used in bioanalysis and biomedical research, construction of these dual-labeled oligonucleotides is relatively laborious, costly and time-consuming. Furthermore, the selectivity or binding affinity of the functional oligonucletide may also be interfered by the bulky fluorophore or quencher group.15 To solve the problem of complex need for duallabeling, various label-free MBs were developed and constructed for DNA, RNA or DNA-RNA hybrid sensors.16-23 Nevertheless, some of them still required complex chemical synthesis and special DNA bases such as the chemical fabrication of luminescent silver nanoclusters with cytosinerich oligonucleotide as the template.24,25 Despite some other label-free MBs have been developed avoiding the steps of chemical synthesis, there still existed a necessary step of separating the recognition strand from the signal probes before the assay step, which was sophisticated and time-consuming, resulting in the limitations in real applications, such as in situ detection.16,17 Therefore, it is highly desirable to develop labeland separation-free MB probes for their real applications. G-quadruplexes, a kind of highly ordered nucleic acid structure with stacked planar G-tetrads,26 have gained a constant attention in the past few decades due to its peroxidase-

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mimicking DNAzyme characteristics after binding to hemin (iron(III)-protoporphyrin IX), the cofactor of natural peroxidase.27 Due to their unique structures, they has been justified as a prospective probe in both positive and negative transcriptional regulation, telomere maintenance and anti-tumor chemotherapy.28-30 In addition, this DNAzyme has been extensively employed as a signal indicator to detect various kinds of targets, such as metal ions, small molecules, nucleic acids, proteins and living cells.31-34 Very recently, some small fluorogenic molecules have been demonstrated to selectively bind to G-quadruplexes resulting in a great fluorescent enhancement, leading to the development of G-quadruplex based fluorescent sensors.31,32 Among these small molecules, Thioflavin T (3,6-dimethyl-2-(4dimethylaminophenyl) benzo-thiazolium cation) has been justified as a novel prospective fluoregenic dye. The free Thioflavin T was composed of two main parts, the benzothiazole (BZT) and dimethylaminobenzene (DMAB). Once upon excitation, the quick rotation of the carbon-carbon single bond between the BZT and DMAB rings could quench Thioflavin T radiative transition in favor of a nonradiative twisted internal charge-transfer (TICT) state; when the dihedral angle at 90°, this TICT effect is especially noticeable. It has already been demonstrated that ThT can induce G-rich oligonucleotide sequences to fold into quadruplexes and sense the quadruplexes motif through fluorescence light-up in a visible region.35 Highly specific fluorescence enhancement of ThT has shown in quadruplex structures compared with the single/double-stranded DNA forms.35,36 Recently due to its low background fluorescence, highly analyte dependence and low cost, various ThT based fluorescent biomolecules and metal ions biosensors were reported. For instance, Tan and coworkers reported a simple approach for specific detection, e.g., DNA, RNA or aptamer.35 However, although a limited number of ThT based label-free fluorescent biosensors have been fabricated, it should be noted that construction of different kinds of Thioflavin T based biosensors is still highly vital due to fact that ThT boosts the characteristics of high sensitivity for its obvious fluorescence enhancement and low cost for its free of modified DNA probe. In this work, we take T4 Polynucleotide kinase (PNK) as an enzyme model using Thioflavin T as a signal producer to evaluate the enzyme activity and its inhibition. T4 Polynucleotide kinase, an important end-processing enzyme with 5’-kinase and 3’-phosphatase activities, is capable of catalyzing the nucleic acid termini converting 5’-OH or 3’-PO4 to 5’-PO4 or 3’-OH, serving as an vital element in catalyzing the phosphorylation process of DNA with 5’-hydroxyl termini in a variety of normal cellular activities, such as DNA recombination, DNA replication and repairing of DNA during the period of strand interruption.51-54 In comparison to the reported T4 PNK assays, besides the high sensitivity, this strategy does not require any labeling and separating process exhibiting a great ease for its only one-step “signal-on”

detection process, which may provide an alternative for T4 PNK assay.

2.Experimental Section 2.1. Chemicals and Materials HPLC-purified oligonucleotides (sequences are listed in Table 1) and λexonuclease (10 U µL-1), were obtained from Sangon Biotechnology Co. Ltd. (Shanghai, China). Thioflavin T (3,6dimethyl-2-(4-dimethylaminophenyl) benzo-thiazolium cation) was bought from Chengdu Xiya Chemical Reagent Co. Ltd (98%) (Chengdu,China). T4 Polynucleotide kinase (PNK) (10 U mL-1), Tris (hydroxymethyl) aminomethane (Tris), adenosine triphosphate (ATP) and other reagents were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All solutions were prepared with ultrapure water (18.2 MΩ cm1) processed by PSDK2-10-C (Beijing, China) apart from the stock solution of hemin was prepared in DMSO and stored in the dark at −20 °C.

TMB

5’-CCTAACCCTTTCTTTCTTTTCAGGG TTAGGGTTAGGGTTAGGG-3’

Table 1 DNA sequences used in this experiment. 2.2. Apparatus Fluorescence spectra were determined by using a Hitachi F4500 fluorescence spectrometer (Hitachi Ltd., Japan) controlled by FL Solution software. The optical path length of a quartz fluorescence cell was 1.0 cm. Excitation and emission slits were all set for a 10.0nm band-pass. The mixtures in square quartz cuvettes were excited at 412nm, and the emission spectra were collected from 450 to 600 nm. The fluorescence intensity at 513nm was used to evaluate the performances of the proposed assay strategy. Absorption spectra were recorded on a UV-via spectrophotometer (Hitachi, Japan) equipped with a quartz cell (1 cm×0.33 cm cross-section) at room temperature. 2.3. Preparation of self-assembled DNA probe stock solution All oligonucleotides were dissolved in 20 mM Tris-HCl buffer (pH=7.4) as stock solutions. They were heated at 90 o C for 10 min and then cooled down to the room temperature. The concentration of the DNA stock solutions were estimated by UV absorption using published sequence-dependent absorption coefficients. The obtained DNA solutions were stored at 4 oC for further use. 2.4. Fluorescent T4 PNK assays In a typical phosphorylation and cleavage assay, 1 µM DNA probe, 0.1 mM ATP, 10 units of λexo and a certain amount of T4 PNK were put into 100 µL of reaction buffer, incubated at 37 oC for 30 mins, and then stopped the cleavage reaction by heating the reaction solution to 85 o C. 20 mM potassium ion and 6 µM ThT were then added to the reaction substrate and

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kept at room temperature for at least 15 min before fluorescence measurement with the excitation wavelength of 412 nM. Control experiments were carried out with ThioflavinT based Molecular Beacon (TMB) replaced by control DNA under the same conditions. 2.5 Charaterization of the digestion process The digestion process was characterized by colorimetry. First, the phosphorylation and cleavage assay were the same as the part 2.4 with no modification. After that, 20 mM K+ and 800 nM hemin were added into the solution after phosphorylation and cleavage and incubated for 2 h to form the hemin Gquadruplex-DNAzyme. Finally, colorimetric system containing 3, 3’, 5, 5’-tetramethylbenzidine and 30 µL 2 mM H2O2 were carried out for the characterization. 2.6 Optimization of analysis conditions In order to obtain the optimal results, the experimental conditions, including the digestion reaction time, concentration of ATP, λexo, K+ , thT, pH value and temperature were evaluated. 2.7 Inhibition assay

2.8 Real samples analysis Human serum samples were obtained from the Yijishan Hospital (Wuhu, China). Before use, the human serum samples were centrifuged at 10 000 rpm for 15 min, and following this, the supernatant was diluted with Tris–HCl (pH 7.4). The analysis process was similar with the fluorescent T4 PNK assays.

3.1. Principle of the sensor for T4 PNK activity

A schematic illustration about the analysis of the T4 PNK activity was shown in Scheme 1. The functionalized TMB probe was composed of three regions: 5’-end with a hydroxyl group for phosphorylation (blue color) and 3’-end (red color) as a blocker of the stem region, and the loop region (black color). As is shown in Scheme 1, in the presence of T4 PNK, the phosphorylation process could happen, allowing the digestion of the 5’-end by λexo and elegantly the λexo is arrested at the linker region due to its inablity to accept single-stranded DNA as a substrate. Thus the 3’-guanine-rich repeats, which is unaffected by the digestion, released and folded into a Gquardruplex. The existing G-quadruplex solution can be recognized by the Thioflavin T dyes. Therefore, the as-formed G-quadruplex can serve as an efficient restricting factor to limit the single C-C bond rotation and enforce planarization of the ThT dye resulting in an enhancement in fluorescence intensity which could quantify the concentration of T4 PNK in a labelfree, effective, convenient and sensitive manner. 3.2. Feasibility investigation of the assay

Different concentrations of Na2HPO4, (NH4)2SO4 and EDTA were added to the phosphorylation and cleavage solution and the steps were similar with the part 2.4.

3. Results and discussion

Scheme 1 Schematic illustration for the detection of T4 PNK activity based on the λexo cleavage reaction and GQuardruplex-ThT complex fluorescence

A set of experiments was carried out to verify the feasibility of our proposed strategy. Fig. 1A shows the fluorescent emission spectra of the probe both in the absence (curve a) and presence (curve b) of T4 PNK. As is demonstrated in Fig. 1A, the fluorescence emission intensity was quite low in the absence of T4 PNK at 513 nm in pH 7.4 Tris-HCl buffer (curve a). It was just as expected that the absence of T4 PNK cannot trigger the phosphorylation process and the λexo catalyzed cleavage reaction. While in the presence of 10 U mL-1 T4 PNK, a fluorescent enhancement appeared as curve b witnessed which confirmed the phosphorylation process helping the happening of the cleavage reaction. Here the wavelength of the maximum emission was slightly different from the previous work reported by Mohanty.36 We hypothesized that the main reason for this wavelength-shifting phenomenon was there were 12-bases overhanging at the end of the remaining oligonucleotides whose aromatic rings would have a steric hindrance effect on the Thioflavin-T-G-quardruplex complexes, thus influencing the ThT’s carbon-carbon bond rotation. In addition, the experimental feasibility was confirmed using a control agarose gel electrophoresis. The result was shown in Fig. 1B. It was observed that in the absence of T4 PNK, a bright band was obtained. When the hairpin DNA was treated in the presence of T4 PNK, no obvious band was observed, indicating a high efficiency of phosphorylation-induced digestion process. The control experiment jointly revealed that T4 PNK played a vital role in catalyzing the phosphorylation process. To confirm the process of TMB probe digested by λexo, the colorimetric method to characterize the digesting products, G-quardruplex sequence was carried out by DNAzyme triggerred the colorimetry of 3, 3’, 5, 5’-tetramethylbenzidine, as shown in Fig. 1 C. In the presence of T4 PNK, the solution shows blue colour and also a characteristic peak of 3, 3’, 5, 5’-

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tetramethylbenzidine (oxidation product) in UV-vis spectra, indicating the formation of the G-rich sequence, which is the product of the digestion. In the absence of T4 PNK, the solution was colourless and there was an unobvious absorbance. Taking advantages of the present strategy, therefore, a simple but effective strategy for T4 PNK assay can be established. 400 350

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intensity was only just the background, while the amounts of λexo was increased to 10 U, the fluorescence intensity achieved the peak. The concentration of K+ and ThT was also investigated, the result in Fig 2D and E. They revealed the amount of K+ had little influence on the fluorescence intensity, indicating the validity of ThT/G-quadruplex based “molecularrotor” mechanism, while the optimal ThT concentration was 6 µM. Therefore 30 min incubation time, 0.1 mM ATP, 10 U λexo, 20 mM K+ and 6 µM ThT were chosen as the optimal conditions. Finally, we examined the effects of experimental conditions including temperature and pH value and the results showed that pH 7.4 and 37 oC were the optimal pH value and the temperature due to the favorable of enzymatic activity.

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Fig. 1 (A) Fluorescence spectra of obtained ThT in the absence (a) and presence (b) of T4 PNK. The concentrations of λexo, T4 PNK, TMB probe and Thioflavin T were 10 U, 10 UmL-1, 100 nM and 6 µM, respectively. (B) Agarose gel (5%) electrophoresis images of TMB in the presence T4 PNK (a) and in the absence of T4 PNK (b). The concentrations of λexo, T4 PNK and TMB were 10U, 10U mL-1 and 1 µM , respectively. (C) Characterization of the digestion process by UV-vis absorption spectra with photographs in the inset of H2O2mediated oxidation of 3, 3’, 5, 5’-tetramethylbenzidine by reaction product in Tris buffer (pH 7.4) in the presence (a) and absence (b) of the T4 PNK. 3.3 Optimization of the Thioflavin T-based T4 PNK assay In order to achieve an optimal experimental result, we investigated the effect of various experimental conditions on the fluorescent intensity in Fig.2. The time of coupled phosphorylation and λexo cleavage reaction plays an critical role in this experiment. In Fig. 2A, as the time of incubation was prolonged, the fluorescence intensity intensified and achieved the maximum in the time of 30 min. Also, the reaction substrate ATP was dispensable for the T4 PNK catalyzed phosphrylation process, therefore we hypothesized that the concentration of ATP was relevant to the value of the final fluorescence intensity. As shown in the Fig. 2B, the relative lower concentration was preferred, because when the 0.1 mM, a slight decrease was observed, concentration of ATP ≧ which implied that higher concentration of ATP may prevent the reaction between ATP and hairpin DNA. Apart from time and reactant of the coupled phosphorylation and cleavage reaction, the catalyst, λexo, was also vital to the sensor. Just as we have expected, when there was no λexo, the fluorescence

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Fig. 3 Inhibitors’ effects of Na2HPO4 (A), (NH4)2SO4 (B) and EDTA (C) on phosphorylation. The assays were carried out in 20 mM Tris–HCl buffer (pH 7.4), containing 10 U mL-1 T4 PNK. 3.5. Detection of T4 PNK and Real sample Application To test the practicality of the proposed sensor, human serum samples were collected as an example and the as-analyzed result was listed and compared with other reported methods in Table 3. The result was satisfying and shown that this method has great potential in real applications.

In summary, we have developed a novel fluorescent sensing system for sensitive detection of T4 PNK activity and its inhibition with very high sensitivity and low detection limit down to 0.001 U mL-1. This fluorescent strategy for T4 PNK activity and its inhibitors boosted the following advantages: (1) This method is label-free, thus it is cheap and simple with only “one-step” detection of T4 PNK . (2) The designed TMB exhibited a low fluorescent background for the detection of T4 PNK. (3) This approach requires no separation of the signal Number

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To evaluate the influences of several inhibitors on the T4 PNK detection, we carried out a set of experiments to investigate the validity of T4 PNK with its inhibitors at different concentrations in the reaction buffer, such as Na2HPO4, EDTA and (NH4)2SO4. It showed that the fluorescence intensity reduced with the increasing concentration of inhibitors in the reaction buffer (Figure 3), which suggested that these three inhibitors were able to deactivate the enzymatic activity of T4 PNK. Herein, there are two main possible reasons for the explanation of the salt effect on kinase activity. Firstly, at high salt concentrations, DNA structure is known to be more stable, which probably prevents the converting process of the 5’hydroxyl group. Secondly, the enzyme conformation may also be significantly affected by a high concentration of salts. The conformational change of the enzyme may further reduce the activity of T4 PNK as well as the affinity between kinase and its substrates. These results indicate that the proposed strategy is promising in quantitatively monitoring the activity of the kinase inhibitors.

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The assays were all carried out in the reaction buffer, containing 1µM TMB and 10 U mL-1 T4 PNK. (S/N = 3)

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generation strand, thus boosting the potential to apply for real sample detection. This simple, cheap, label and separation-free method for the detection of T4 PNK may provide an alternative in the real sample analysis which may contribute to the monitoring of the phosphorylation process of DNA in various normal cellular activities.

Acknowledgements This work was financially supported by the projects (No. 21371007) from National Natural Science Foundation of China, Anhui Provincial Natural Science Foundation (1208085QB28), Anhui Provincial Natural Science Foundation for Distinguished Youth (1408085J03), Natural Science Foundation of Anhui (KJ2012A139) and the Program for Innovative Research Team at Anhui Normal University.

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One-strand oligonucleotide probe for fluorescent label-free "turn-on" detection of T4 polynucleotide kinase activity and its inhibition.

Thioflavin T (ThT), as one of the most exciting fluorogenic molecules, boasts the "molecular-rotor" ability to induce DNA sequences containing guanine...
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