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Yuecheng Zhang, Chenghui Liu,* Sujuan Sun, Yanli Tang and Zhengping Li* 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x A versatile flow cytometric bead assay (FCBA) is developed for ultrasensitive detection of T4 PNK activity by integrating the advantages of phosphorylation-induced hybridization chain reaction (HCR) for fluorescence signal amplification and flow cytometry for robust, sensitive and rapid signal readout of the microbeads (MBs). T4 polynucleotide kinase (T4 PNK), a crucial enzyme that catalyzes the phosphorylation of nucleic acid with 5'-hydroxyl termini, plays critical roles in a majority of cellular events.1,2 As is well-recognized that many genotoxic agents can induce DNA strand breaks generally associated with the generation of 5'hydroxyl termini. The phosphorylation of such damaged 5'termini by T4 PNK is the prerequisite for ligation-based healing of DNA lesions to maintain gene integrity.1 It has been revealed that abnormal T4 PNK activity may be closely associated with some serious human disorders such as Bloom’s syndrome, Rothmund-Thomson syndrome and Werner syndrome.3 Therefore, on account of these important biological and clinical roles of T4 PNK, the development of sensitive assays for assessing T4 PNK activity has attracted a lot of interest and has become a popular topic in recent years. Traditionally, T4 PNK-catalyzed DNA phosphorylation used to be analyzed by radioactive 32P-labeling, polyacrylamide gel electrophoresis (PAGE) and autoradiography.2, 4-5 To overcome the inherent drawbacks of these methods such as complicated procedures and potential radioactive hazard to human health, in recent years, great efforts have been made towards the development of nonradioactive T4 PNK assays. Up to now, various techniques including fluorescence,6 electrochemistry,7 colorimetric strategy8 and chemi/bio-luminescence9 have been developed for the detection of T4 PNK activities by combining with either functional nucleic acid probes (e.g., molecular beacons, DNAzymes) or functional nanomaterials (e.g., graphene oxide, TiO2, Au nanoparticle). Among these recently developed T4 PNK assays, the homogeneous fluorescent strategies are the most popular and attractive due to their separation-free characteristics, design flexibility and easy signal readout. However, it should be noted that most of these homogeneous fluorescent assays involve the cuvette-based measurements with a fluorometer. An intrinsic limitation is that when challenged with clinical biosamples with cloudy optical appearance, the strong This journal is © The Royal Society of Chemistry [year]
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light scattering and autofluorescence of the complex biological matrix will greatly interfere with the fluorescence detection.10 Therefore, despite the great progresses being made for T4 PNK assays, further improvement of the analytical performances, particularly sensitivity and high tolerant capability for complex biological matrices, is still in urgent demand for T4 PNK-related biological research, clinic diagnostics, and drug discovery. Herein, we wish to report a robust flow cytometric bead assay (FCBA) for ultrasensitive detection of T4 PNK activity by use of hybridization chain reaction (HCR) for fluorescence signal amplification on the bead surface and flow cytometry for powerful signal readout of the microbeads (MBs).
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Fig. 1 Schematic illustration of the proposed FCBA for the detection of T4 PNK activity.
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The work principle of the proposed FCBA is illustrated in Fig. 1. Firstly, a biotinylated HCR Initiator (sequence 1-2) with an additional ten thymine nucleotides (T10) at its 5'-terminus is immobilized on the streptavidin-functionalized magnetic MBs (STV-MBs) via the specific STV-biotin interaction. The T10 sequence is introduced as a spacer to ensure high accessibility of enzymes and HCR fuels to the DNA sequences anchored on the MBs surface. Then the HCR Initiators on the MBs are completely blocked with the complementary Blocking DNA sequences. In addition, two species of fluorescein (FAM)-labeled stem-loop DNA hairpin (H1 and H2) are rationally designed as the HCR fuels, which can stably coexist in the solution in the absence of HCR Initiator.11 Under the catalysis of T4 PNK, the γ-phosphoryl of ATP will be transferred to the 5'-OH terminus of Blocking DNA. It is well-recognized that λ exonuclease (λ exo) is a 5'→3' exodeoxyribonuclease that can specifically digest the 5'-PO4 end of double stranded DNA (dsDNA) at a high speed.12 Therefore, [journal], [year], [vol], 00–00 | 1
ChemComm Accepted Manuscript
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Phosphorylation-Induced Hybridization Chain Reaction on Beads: An Ultrasensitive Flow Cytometric Assay for the Detection of T4 Polynucleotide Kinase Activity
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Fig. 2 (a) Scatter plots (FL1 vs. FSC) of the FCBA system in the presence of different concentrations of T4 PNK. 10000 MBs were collected for each sample; (b) fluorescence imaging results of the DNA-MBs treated with 0 (blank control), 0.0001, and 0.001 U/mL T4 PNK. The left images are fluorescence images and the right ones are bright field images. 5
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the 5'-phosphorylated Blocking DNA would be recognized and efficiently digested by λ exo, which in turn liberates the blocked HCR Inhibitor sequence. In this regard, upon the addition of H1 and H2, the freed HCR Inhibitor sequence (1-2) will pair with the sticky end of H1 (1*-2*) and open its hairpin through an unbiased strand-displacement reaction. Subsequently, the newly released sticky end of H1 (3-2) on the MBs will further hybridize with the sticky end of H2 (3*-2*) to open the H2 hairpin and further expose a new single stranded sequence (1-2). This sequence is identical to the HCR Initiator, which will in turn trigger a new cycle of hybridization between H1 and H2. In this manner, each liberated HCR Initiator on the MBs will initiate a cascade chain reaction of hybridization events between alternating H1 and H2, resulting in greatly amplified accumulation of fluorophores on the MBs. In contrast, if T4 PNK is absent, the unphosphorylated Blocking DNA with 5'-OH terminus will not be cleaved by λ exo, which will prevent the HCR Initiator from triggering HCR due to the efficient blocking. Finally, in this design, the fluorescence signals of the MBs are directly analyzed as they pass one-by-one through a flow cytometer, which enables sequential detection of individual MBs at a high speed of up to thousands of beads per second. Through statistically analyzing numerous fluorescence data collected from 10000 MBs for each sample, T4 PNK activities will be quantitatively reflected by the fluorescence signal of HCR products anchored on the MBs. This strategy exhibits several distinct advantages. Firstly, the efficient HCR signal amplification mechanism, which will be initiated only upon the T4 PNK-catalyzed DNA phosphorylation This journal is © The Royal Society of Chemistry [year]
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and the sequential λ exo cleavage reaction (see Fig. S2), is utilized for fluorescence amplification and enrichment on the MBs, giving rise to a significantly enhanced sensitivity. Secondly, this MBs-based assay allows easy abstraction of the fluorescence background from the sample matrices via magnetic isolation. Meanwhile, flow cytometry is well suited for highly sensitive quantitation of the MBs fluorescence in complex mixtures because flow cytometer itself can automatically discriminate the interfering fluorescence signals from those of MBs with a given size.10,13 As a result, the fluorescence background of the reaction system can be significantly eliminated. Therefore, by integrating these advantageous features, we have achieved the highest sensitivity known so far for T4 PNK analysis. Under the systematically optimized experimental conditions such as the amount of ATP and λ exo (ESI), the analytical performance of the FCBA for the detection of T4 PNK activity has been investigated. Analytical flow cytometry is a powerful technique for MBs analysis which enables multiple parameters signal readout including the fluorescence and light scattering properties of the MBs. As we know, the forward scatter (FSC) signal is independent of the bead fluorescence but only determined by the size and shape of the detected MBs. Fig. 2a shows the scatter plots (FL1 vs. FSC) of the FCBA system when different concentrations of T4 PNK are introduced. Since the MBs are monodisperse and uniform in sizes, one can see that the FSC values of all MBs populations remain stable despite of the different T4 PNK activities. So the MBs can be easily discriminated from the matrix background through their [journal], [year], [vol], 00–00 | 2
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excellent specificity towards T4 PNK.
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Fig. 3 (a) Histograms of fluorescence response of the FCBA system in the presence of different concentrations of T4 PNK. T4 PNK from left to the right: 0 (black), 0.00001 (red), 0.00005 (blue), 0.0001 (green), 0.0005 (orange), 0.001 (purple), 0.01 (pink), 0.1 (brown), 1 U/ml (dark yellow), respectively; (b) relationship between the MFI values and T4 PNK activities. (inset) calibration curve between MFI and T4 PNK activity in the range from 0.00001 to 0.001 U/ml. The error bars represent standard deviation of three replicates for each data point.
Furthermore, a practical bioassay should be applicable for target analysis in complex biological samples. To evaluate the robustness of the FCBA, we further investigate its analytical performance for the detection of T4 PNK activity by using DMEM cell culture medium as the model complex biological fluids. The DMEM samples (4 µL, 20% of the total 20 µL reaction volume), which were spiked with varying concentrations of T4 PNK, were detected by the FCBA. The results shown in Fig. S5 suggest that the proposed FCBA works well in the complex DMEM matrix. The MFI increases gradually with the increase of spiked T4 PNK. More fascinatingly, it can be also found that when using DMEM as the matrix, the MFI values produced by different T4 PNK activities are similar to those acquired in clean buffer. Therefore, it is possible to quantitatively evaluate the T4 PNK activity in complex biosamples by using the simultaneously constructed calibration curve in clean buffer system. It has been reported that inhibition of PNK activity may potentially increase the sensitivity of tumors to γ-radiation,14 so PNK inhibitors may become potential drugs to enhance the efficacy of traditional cancer treatment. Therefore, we have conducted further experiments to test the feasibility of this FCBA for screening of potential T4 PNK inhibitors. Three known T4 PNK inhibitors ((NH4)2SO4, Na2HPO4 and ADP), which have been proved to have no influence on λ exo activity,6b, 15 are used in this inhibition study by fixing T4 PNK at 0.01 U/mL. Fig. 4 shows the inhibition effect of (NH4)2SO4 on T4 PNK. As can be Journal Name, [year], [vol], 00–00 | 3
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characterized FSC signal. Although the FSC signal is constant, Fig. 2a shows that the fluorescence signal of the MBs detected in FL1 (FAM) channel exhibits a clear elevation tendency with the increase of T4 PNK activity. One can see that for the blank control without T4 PNK, all of the MBs are concentrated in a small region. However, when low concentrations of T4 PNK is introduced (0.00001~0.0005 U/mL), the MBs of each sample are divided into two populations, one of which is highly fluorescent while the other is not. The reason may be that in such cases, the number of DNA-functionalized MBs (DNA-MBs) may greatly exceed the catalytic capacity of T4 PNK, so the phosphorylation of the Blocking DNA and the subsequent λ exo digestion and HCR amplification can only occur on a portion of MBs, and only these MBs are enriched with fluorescent HCR products while the other DNA-MBs will not. With the increase of T4 PNK activity, more and more DNA-MBs will participate in the reaction and thus will become highly fluorescent. The phenomena are well consistent with the fluorescence imaging results. As shown in Fig. 2b, no fluorescent MBs are observed for the blank control while about half of the MBs are bright at T4 PNK activity of 0.0001 U/mL. When further increasing T4 PNK to 0.001 U/mL, almost all MBs become intensively bright. Fig. 3a displays the corresponding fluorescence histograms of the MBs, from which the T4 PNK dose-responsive signal increase in FL1 channel can be clearly observed. The mean fluorescence intensities (MFI) of all the 10000 detected MBs for each sample are used for the quantitative analysis of T4 PNK activity, and the relationship between the MFI values and T4 PNK activities is plotted in Fig. 3b. It can be seen that the MFI is linearly proportional to the T4 PNK activity in the range from 0.00001 to 0.001 U/mL, and as low as 0.00001 U/ml T4 PNK can be unequivocally detected. The correlation equation is MFI=1.27×105+1.52×109CT4 PNK (U/mL, R=0.9987), and the corresponding detection limit (3σ) is calculated to be 4×10-6 U/ml. As far as we know, the recently developed electrochemical and fluorometric methods coupled with either functional nanomaterials or DNA probes are the most popular and sensitive assays for T4 PNK.6,7 The detection limits of T4 PNK by these protocols generally fall in the range of 0.001~0.05 U/mL. Notably, an exceptional result is reported by Zhao group more recently.6e With the ligase/nicking enzyme-assisted nucleic acid amplification, 0.00002 U/mL T4 PNK can be detected in their work. Therefore, one can see that the sensitivity of our FCBA strategy is at least two orders of magnitude higher than those of most existing T4 PNK assays, and is also superior to that of Zhao group’s protocol. To the best of our knowledge, the detection limit of T4 PNK (4×10-6 U/mL) by using the proposed FCBA is the lowest thus far for T4 PNK analysis. Besides for the high sensitivity, specificity is another important aspect to evaluate a bioassay. To investigate the specificity of this proposed method for T4 PNK, the FCBA system is further challenged with several other proteins including protein kinases (PKA and Src), hexokinase (HK), bovine serum albumin (BSA), T4 DNA ligase as well as heat-inactivated T4 PNK, respectively. As can be seen from the results shown in Fig. S4, only T4 PNK arouses remarkable increase of MFI while all of the other targets hardly generate any responses compared with the blank control. These results demonstrate that the proposed FCBA exhibits
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seen that the MFI values decrease gradually along with the increase doses of (NH4)2SO4, clearly suggesting the effective inhibition of T4 PNK activity. Similarly, as displayed in Fig. S6, increasing concentrations of Na2HPO4 or ADP both result in the gradual decrease of MFI. The addition of approximately 15 mM (NH4)2SO4, 20 mM Na2HPO4 or merely 0.07 mM ADP can effectively suppress the T4 PNK activity around 50%. These results indicate that the developed FCBA can be applicable for screening of potential T4 PNK inhibitor drugs.
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Fig. 4 Inhibition effects of (NH4)2SO4 on T4 PNK activity (fixed at 0.01 U/mL). (inset) the plot between the MFI values and the concentrations of (NH4)2SO4. Other conditions: λ exo, 0.1 U/ml; ATP, 1 mM.
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In summary, a flow cytometry-based assay, which consists of a series of cascade reactions including sequentially T4 PNKcatalyzed DNA phosphorylation, phosphorylation-activated DNA hydrolysis by λ exo, and DNA digestion-actuated HCR signal amplification, is rationally designed and conducted on the surface of MBs for the ultrasensitive and selective detection of T4 PNK activity. Due to the greatly amplified fluorophore accumulation on MBs through HCR and the powerful flow cytometry for rapid and quantitative bead signal readout, excellent analytical performance is acquired for the proposed FCBA with an extremely low detection limit of 4×10-6 U/mL of T4 PNK. Considering the high sensitivity, selectivity and high tolerant capability for complex biological matrices, we believe that the FCBA is of great potential for the applications in PNK-related biological process research, drug discovery, and diagnostics. This work was supported by the National Natural Science Foundation of China (21335005, 21472120), Program for Innovative Research Team in Shaanxi Province (No. 2014KCT28), the Natural Science Foundation of Shaanxi Province (2014JQ2058), the Fundamental Research Funds for the Central Universities (GK201402051, GK201303003), and the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201409).
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† Electronic Supplementary Information (ESI) available: [Detailed Experimental procedures, optimization of experimental conditions, T4 PNK analysis in complex samples and the inhibition effect of Na2HPO4 and ADP on T4 PNK activity]. See DOI: 10.1039/b000000x/ 1 L. K. Wang, C. D. Lima and S. Shuman, EMBO J., 2002, 21, 3873. 2 C. J. Whitehouse, R. M. Taylor, A. Thistlethwaite, H. Zhang, F. Karimi-busheri, D. D. Lasko, M. Weinfeld and K. W. Caldecott, Cell, 2001, 104, 107. 3 S. Sharma, K. M. Doherty and R. M. Brosh, Biochem. J., 2006, 398, 319. 4 M. Meijer, F. Karimi-Busheri, T. Y. Huang, M. Weinfeld and D. Young, J. Biol. Chem., 2002, 277, 4050. 5 L. K. Wang and S. Shuman, J. Biol. Chem., 2001, 276, 26868. 6 (a) L. Lin, Y. Liu, X. Zhao and J. Li, Anal. Chem., 2011, 83, 8396; (b) T. Hou, X. Wang, X. Liu, T. Lu, S. Liu and F. Li, Anal. Chem., 2014, 86, 884; (c) C. Song, X. Yang, K. Wang, Q. Wang, J. Liu, J. Huang, L. He, P. Liu, Z. Qing and W. Liu, Chem. Commun., 2015, 51, 1815; (d) S. Liu, J. Ming, Y. Lin, C. Wang, C. Cheng, T. Liu and L. Wang, Biosens. Bioelectron., 2014, 55, 225; (e) F. Chen, Y. Zhao, L. Qi and C. Fan, Biosens. Bioelectron., 2013, 47, 218. 7 (a) Y. Peng, J. Jiang and R. Yu, RSC Adv., 2013, 3, 18128; (b) Y. Wang, X. He, K. Wang, X. Ni, J. Su and Z. Chen, Biosens. Bioelectron., 2012, 32, 213; (c) G. Wang, X. He, G. Xu, L. Chen, Y. Zhu, X. Zhang and L. Wang, Biosens. Bioelectron., 2013, 43, 125. 8 H. Jiang, D. Kong and H. Shen, Biosens. Bioelectron., 2014, 55, 133. 9 (a) H. He, K. Leung, W. Wang, D. Chan, C. Leung and D. Ma, Chem. Commun., 2014, 50, 5313; (b) J. Du, Q. Xu, X. Lu and C. Zhang, Anal. Chem., 2014, 86, 8481; (c) W. Tang, G. Zhu and C. Zhang, Chem. Commun., 2014, 50, 4733. 10 P.-J. J. Huang and J. Liu, Anal. Chem., 2010, 82, 4020. 11 R. M. Dirks and N. A. Pierce, Proc. Natl. Acad. Sci. U.S.A., 2004, 101, 15275. 12 (a) J. W. Little, J. Biol. Chem., 1967, 242, 679; (b) R. G. Higuchi and H. Ochman, Nucleic Acid Res., 1989, 17, 5865. 13 (a) D. Nie, H. Wu, Q. Zheng, L. Guo, P. Ye, Y. Hao, Y. Li, F. Fu and Y. Guo, Chem. Commun., 2012, 48, 1150; (b) W. Ren, H. Liu, W. Yang, Y. Fan, L. Yang, Y. Wang, C. Liu and Z. Li, Biosens. Bioelectron., 2013, 49, 380; (c) W. Ren, C. Liu, S. Lian and Z. Li, Anal. Chem., 2013, 85, 10956. 14 (a) T. R. Mereniuk, R. A. Maranchuk, A. Schindler, J. Penner-Chea, G. K. Freschauf, S. Hegazy, R. Lai, E. Foley and M. Weinfeld, Cancer Res., 2012, 72, 5934; (b) A. Rasouli-Nia, F. Karimi-Busheri and M. Weinfeld, Proc. Natl. Acad. Sci. U.S.A., 2004, 101, 6905. 15 R. S. Swathi and K. L. Sebastian, J. Chem. Phys., 2009, 130, 086101.
Notes and references
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Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, Shaanxi Province, P. R. China. Tel/Fax: +86 29 81530859; E-mail: [email protected]; [email protected]
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This journal is © The Royal Society of Chemistry [year]
ChemComm Accepted Manuscript
DOI: 10.1039/C5CC00572H
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Y. Zhang, C. Liu, S. Sun, Y. Tang and Z. Li, Chem. Commun., 2015, DOI: 10.1039/C5CC00572H.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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DOI: 10.1039/C5CC00572H
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Yuecheng Zhang, Chenghui Liu,* Sujuan Sun, Yanli Tang and Zhengping Li* 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x A versatile flow cytometric bead assay (FCBA) is developed for ultrasensitive detection of T4 PNK activity by integrating the advantages of phosphorylation-induced hybridization chain reaction (HCR) for fluorescence signal amplification and flow cytometry for robust, sensitive and rapid signal readout of the microbeads (MBs). T4 polynucleotide kinase (T4 PNK), a crucial enzyme that catalyzes the phosphorylation of nucleic acid with 5'-hydroxyl termini, plays critical roles in a majority of cellular events.1,2 As is well-recognized that many genotoxic agents can induce DNA strand breaks generally associated with the generation of 5'hydroxyl termini. The phosphorylation of such damaged 5'termini by T4 PNK is the prerequisite for ligation-based healing of DNA lesions to maintain gene integrity.1 It has been revealed that abnormal T4 PNK activity may be closely associated with some serious human disorders such as Bloom’s syndrome, Rothmund-Thomson syndrome and Werner syndrome.3 Therefore, on account of these important biological and clinical roles of T4 PNK, the development of sensitive assays for assessing T4 PNK activity has attracted a lot of interest and has become a popular topic in recent years. Traditionally, T4 PNK-catalyzed DNA phosphorylation used to be analyzed by radioactive 32P-labeling, polyacrylamide gel electrophoresis (PAGE) and autoradiography.2, 4-5 To overcome the inherent drawbacks of these methods such as complicated procedures and potential radioactive hazard to human health, in recent years, great efforts have been made towards the development of nonradioactive T4 PNK assays. Up to now, various techniques including fluorescence,6 electrochemistry,7 colorimetric strategy8 and chemi/bio-luminescence9 have been developed for the detection of T4 PNK activities by combining with either functional nucleic acid probes (e.g., molecular beacons, DNAzymes) or functional nanomaterials (e.g., graphene oxide, TiO2, Au nanoparticle). Among these recently developed T4 PNK assays, the homogeneous fluorescent strategies are the most popular and attractive due to their separation-free characteristics, design flexibility and easy signal readout. However, it should be noted that most of these homogeneous fluorescent assays involve the cuvette-based measurements with a fluorometer. An intrinsic limitation is that when challenged with clinical biosamples with cloudy optical appearance, the strong This journal is © The Royal Society of Chemistry [year]
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light scattering and autofluorescence of the complex biological matrix will greatly interfere with the fluorescence detection.10 Therefore, despite the great progresses being made for T4 PNK assays, further improvement of the analytical performances, particularly sensitivity and high tolerant capability for complex biological matrices, is still in urgent demand for T4 PNK-related biological research, clinic diagnostics, and drug discovery. Herein, we wish to report a robust flow cytometric bead assay (FCBA) for ultrasensitive detection of T4 PNK activity by use of hybridization chain reaction (HCR) for fluorescence signal amplification on the bead surface and flow cytometry for powerful signal readout of the microbeads (MBs).
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Fig. 1 Schematic illustration of the proposed FCBA for the detection of T4 PNK activity.
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The work principle of the proposed FCBA is illustrated in Fig. 1. Firstly, a biotinylated HCR Initiator (sequence 1-2) with an additional ten thymine nucleotides (T10) at its 5'-terminus is immobilized on the streptavidin-functionalized magnetic MBs (STV-MBs) via the specific STV-biotin interaction. The T10 sequence is introduced as a spacer to ensure high accessibility of enzymes and HCR fuels to the DNA sequences anchored on the MBs surface. Then the HCR Initiators on the MBs are completely blocked with the complementary Blocking DNA sequences. In addition, two species of fluorescein (FAM)-labeled stem-loop DNA hairpin (H1 and H2) are rationally designed as the HCR fuels, which can stably coexist in the solution in the absence of HCR Initiator.11 Under the catalysis of T4 PNK, the γ-phosphoryl of ATP will be transferred to the 5'-OH terminus of Blocking DNA. It is well-recognized that λ exonuclease (λ exo) is a 5'→3' exodeoxyribonuclease that can specifically digest the 5'-PO4 end of double stranded DNA (dsDNA) at a high speed.12 Therefore, [journal], [year], [vol], 00–00 | 1
ChemComm Accepted Manuscript
Published on 05 February 2015. Downloaded by Gazi Universitesi on 05/02/2015 15:34:06.
Phosphorylation-Induced Hybridization Chain Reaction on Beads: An Ultrasensitive Flow Cytometric Assay for the Detection of T4 Polynucleotide Kinase Activity
Journal Name
ChemComm
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DOI: 10.1039/C5CC00572H
Cite this: DOI: 10.1039/c0xx00000x
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Fig. 2 (a) Scatter plots (FL1 vs. FSC) of the FCBA system in the presence of different concentrations of T4 PNK. 10000 MBs were collected for each sample; (b) fluorescence imaging results of the DNA-MBs treated with 0 (blank control), 0.0001, and 0.001 U/mL T4 PNK. The left images are fluorescence images and the right ones are bright field images. 5
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the 5'-phosphorylated Blocking DNA would be recognized and efficiently digested by λ exo, which in turn liberates the blocked HCR Inhibitor sequence. In this regard, upon the addition of H1 and H2, the freed HCR Inhibitor sequence (1-2) will pair with the sticky end of H1 (1*-2*) and open its hairpin through an unbiased strand-displacement reaction. Subsequently, the newly released sticky end of H1 (3-2) on the MBs will further hybridize with the sticky end of H2 (3*-2*) to open the H2 hairpin and further expose a new single stranded sequence (1-2). This sequence is identical to the HCR Initiator, which will in turn trigger a new cycle of hybridization between H1 and H2. In this manner, each liberated HCR Initiator on the MBs will initiate a cascade chain reaction of hybridization events between alternating H1 and H2, resulting in greatly amplified accumulation of fluorophores on the MBs. In contrast, if T4 PNK is absent, the unphosphorylated Blocking DNA with 5'-OH terminus will not be cleaved by λ exo, which will prevent the HCR Initiator from triggering HCR due to the efficient blocking. Finally, in this design, the fluorescence signals of the MBs are directly analyzed as they pass one-by-one through a flow cytometer, which enables sequential detection of individual MBs at a high speed of up to thousands of beads per second. Through statistically analyzing numerous fluorescence data collected from 10000 MBs for each sample, T4 PNK activities will be quantitatively reflected by the fluorescence signal of HCR products anchored on the MBs. This strategy exhibits several distinct advantages. Firstly, the efficient HCR signal amplification mechanism, which will be initiated only upon the T4 PNK-catalyzed DNA phosphorylation This journal is © The Royal Society of Chemistry [year]
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and the sequential λ exo cleavage reaction (see Fig. S2), is utilized for fluorescence amplification and enrichment on the MBs, giving rise to a significantly enhanced sensitivity. Secondly, this MBs-based assay allows easy abstraction of the fluorescence background from the sample matrices via magnetic isolation. Meanwhile, flow cytometry is well suited for highly sensitive quantitation of the MBs fluorescence in complex mixtures because flow cytometer itself can automatically discriminate the interfering fluorescence signals from those of MBs with a given size.10,13 As a result, the fluorescence background of the reaction system can be significantly eliminated. Therefore, by integrating these advantageous features, we have achieved the highest sensitivity known so far for T4 PNK analysis. Under the systematically optimized experimental conditions such as the amount of ATP and λ exo (ESI), the analytical performance of the FCBA for the detection of T4 PNK activity has been investigated. Analytical flow cytometry is a powerful technique for MBs analysis which enables multiple parameters signal readout including the fluorescence and light scattering properties of the MBs. As we know, the forward scatter (FSC) signal is independent of the bead fluorescence but only determined by the size and shape of the detected MBs. Fig. 2a shows the scatter plots (FL1 vs. FSC) of the FCBA system when different concentrations of T4 PNK are introduced. Since the MBs are monodisperse and uniform in sizes, one can see that the FSC values of all MBs populations remain stable despite of the different T4 PNK activities. So the MBs can be easily discriminated from the matrix background through their [journal], [year], [vol], 00–00 | 2
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excellent specificity towards T4 PNK.
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Fig. 3 (a) Histograms of fluorescence response of the FCBA system in the presence of different concentrations of T4 PNK. T4 PNK from left to the right: 0 (black), 0.00001 (red), 0.00005 (blue), 0.0001 (green), 0.0005 (orange), 0.001 (purple), 0.01 (pink), 0.1 (brown), 1 U/ml (dark yellow), respectively; (b) relationship between the MFI values and T4 PNK activities. (inset) calibration curve between MFI and T4 PNK activity in the range from 0.00001 to 0.001 U/ml. The error bars represent standard deviation of three replicates for each data point.
Furthermore, a practical bioassay should be applicable for target analysis in complex biological samples. To evaluate the robustness of the FCBA, we further investigate its analytical performance for the detection of T4 PNK activity by using DMEM cell culture medium as the model complex biological fluids. The DMEM samples (4 µL, 20% of the total 20 µL reaction volume), which were spiked with varying concentrations of T4 PNK, were detected by the FCBA. The results shown in Fig. S5 suggest that the proposed FCBA works well in the complex DMEM matrix. The MFI increases gradually with the increase of spiked T4 PNK. More fascinatingly, it can be also found that when using DMEM as the matrix, the MFI values produced by different T4 PNK activities are similar to those acquired in clean buffer. Therefore, it is possible to quantitatively evaluate the T4 PNK activity in complex biosamples by using the simultaneously constructed calibration curve in clean buffer system. It has been reported that inhibition of PNK activity may potentially increase the sensitivity of tumors to γ-radiation,14 so PNK inhibitors may become potential drugs to enhance the efficacy of traditional cancer treatment. Therefore, we have conducted further experiments to test the feasibility of this FCBA for screening of potential T4 PNK inhibitors. Three known T4 PNK inhibitors ((NH4)2SO4, Na2HPO4 and ADP), which have been proved to have no influence on λ exo activity,6b, 15 are used in this inhibition study by fixing T4 PNK at 0.01 U/mL. Fig. 4 shows the inhibition effect of (NH4)2SO4 on T4 PNK. As can be Journal Name, [year], [vol], 00–00 | 3
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characterized FSC signal. Although the FSC signal is constant, Fig. 2a shows that the fluorescence signal of the MBs detected in FL1 (FAM) channel exhibits a clear elevation tendency with the increase of T4 PNK activity. One can see that for the blank control without T4 PNK, all of the MBs are concentrated in a small region. However, when low concentrations of T4 PNK is introduced (0.00001~0.0005 U/mL), the MBs of each sample are divided into two populations, one of which is highly fluorescent while the other is not. The reason may be that in such cases, the number of DNA-functionalized MBs (DNA-MBs) may greatly exceed the catalytic capacity of T4 PNK, so the phosphorylation of the Blocking DNA and the subsequent λ exo digestion and HCR amplification can only occur on a portion of MBs, and only these MBs are enriched with fluorescent HCR products while the other DNA-MBs will not. With the increase of T4 PNK activity, more and more DNA-MBs will participate in the reaction and thus will become highly fluorescent. The phenomena are well consistent with the fluorescence imaging results. As shown in Fig. 2b, no fluorescent MBs are observed for the blank control while about half of the MBs are bright at T4 PNK activity of 0.0001 U/mL. When further increasing T4 PNK to 0.001 U/mL, almost all MBs become intensively bright. Fig. 3a displays the corresponding fluorescence histograms of the MBs, from which the T4 PNK dose-responsive signal increase in FL1 channel can be clearly observed. The mean fluorescence intensities (MFI) of all the 10000 detected MBs for each sample are used for the quantitative analysis of T4 PNK activity, and the relationship between the MFI values and T4 PNK activities is plotted in Fig. 3b. It can be seen that the MFI is linearly proportional to the T4 PNK activity in the range from 0.00001 to 0.001 U/mL, and as low as 0.00001 U/ml T4 PNK can be unequivocally detected. The correlation equation is MFI=1.27×105+1.52×109CT4 PNK (U/mL, R=0.9987), and the corresponding detection limit (3σ) is calculated to be 4×10-6 U/ml. As far as we know, the recently developed electrochemical and fluorometric methods coupled with either functional nanomaterials or DNA probes are the most popular and sensitive assays for T4 PNK.6,7 The detection limits of T4 PNK by these protocols generally fall in the range of 0.001~0.05 U/mL. Notably, an exceptional result is reported by Zhao group more recently.6e With the ligase/nicking enzyme-assisted nucleic acid amplification, 0.00002 U/mL T4 PNK can be detected in their work. Therefore, one can see that the sensitivity of our FCBA strategy is at least two orders of magnitude higher than those of most existing T4 PNK assays, and is also superior to that of Zhao group’s protocol. To the best of our knowledge, the detection limit of T4 PNK (4×10-6 U/mL) by using the proposed FCBA is the lowest thus far for T4 PNK analysis. Besides for the high sensitivity, specificity is another important aspect to evaluate a bioassay. To investigate the specificity of this proposed method for T4 PNK, the FCBA system is further challenged with several other proteins including protein kinases (PKA and Src), hexokinase (HK), bovine serum albumin (BSA), T4 DNA ligase as well as heat-inactivated T4 PNK, respectively. As can be seen from the results shown in Fig. S4, only T4 PNK arouses remarkable increase of MFI while all of the other targets hardly generate any responses compared with the blank control. These results demonstrate that the proposed FCBA exhibits
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seen that the MFI values decrease gradually along with the increase doses of (NH4)2SO4, clearly suggesting the effective inhibition of T4 PNK activity. Similarly, as displayed in Fig. S6, increasing concentrations of Na2HPO4 or ADP both result in the gradual decrease of MFI. The addition of approximately 15 mM (NH4)2SO4, 20 mM Na2HPO4 or merely 0.07 mM ADP can effectively suppress the T4 PNK activity around 50%. These results indicate that the developed FCBA can be applicable for screening of potential T4 PNK inhibitor drugs.
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Fig. 4 Inhibition effects of (NH4)2SO4 on T4 PNK activity (fixed at 0.01 U/mL). (inset) the plot between the MFI values and the concentrations of (NH4)2SO4. Other conditions: λ exo, 0.1 U/ml; ATP, 1 mM.
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In summary, a flow cytometry-based assay, which consists of a series of cascade reactions including sequentially T4 PNKcatalyzed DNA phosphorylation, phosphorylation-activated DNA hydrolysis by λ exo, and DNA digestion-actuated HCR signal amplification, is rationally designed and conducted on the surface of MBs for the ultrasensitive and selective detection of T4 PNK activity. Due to the greatly amplified fluorophore accumulation on MBs through HCR and the powerful flow cytometry for rapid and quantitative bead signal readout, excellent analytical performance is acquired for the proposed FCBA with an extremely low detection limit of 4×10-6 U/mL of T4 PNK. Considering the high sensitivity, selectivity and high tolerant capability for complex biological matrices, we believe that the FCBA is of great potential for the applications in PNK-related biological process research, drug discovery, and diagnostics. This work was supported by the National Natural Science Foundation of China (21335005, 21472120), Program for Innovative Research Team in Shaanxi Province (No. 2014KCT28), the Natural Science Foundation of Shaanxi Province (2014JQ2058), the Fundamental Research Funds for the Central Universities (GK201402051, GK201303003), and the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201409).
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Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, Shaanxi Province, P. R. China. Tel/Fax: +86 29 81530859; E-mail: [email protected]; [email protected]
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ChemComm Accepted Manuscript
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