A Facile and Sensitive Method for Protein Kinase A Activity Assay

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A Facile and Sensitive Method for Protein Kinase A Activity Assay Based on Fluorescent Off-on PolyU-peptide Assembly Yanhui Xu, Wen Shi, Xinyuan He, Xiaofeng Wu, Xiaohua Li, and Huimin Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02815 • Publication Date (Web): 22 Sep 2017 Downloaded from http://pubs.acs.org on September 22, 2017

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Analytical Chemistry

A Facile and Sensitive Method for Protein Kinase A Activity Assay Based on Fluorescent Off-on PolyU-peptide Assembly Yanhui Xu,a,b Wen Shi,*,a,b Xinyuan He,a Xiaofeng Wu,a Xiaohua Li,a Huimin Maa,b a

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. b University of Chinese Academy of Sciences, Beijing 100049, China. ABSTRACT: Phosphorylation mediated by protein kinases plays a pivotal role in metabolic and cell-signaling processes, and the dysfunction of protein kinases such as protein kinase A (PKA) may induce several human diseases. Therefore it is of great significance to develop a facile and effective method for PKA activity assay and high-throughput screening of inhibitors. Herein, we develop a new fluorescent off-on method for PKA assay based on the assembly of anionic polyuridylic acid (polyU) and cationic fluorescent peptide. The phosphorylation of the peptide disrupts its electrostatic binding with polyU, suppresses the concentration quenching effect of polyU and thus causes fluorescence recovery. The recovered fluorescence intensity at 585 nm is directly proportional to the PKA activity in the range of 0.1~3.2 U/mL with a detection limit of 0.05 U/mL. Using our method, the PKA activity in HeLa cell lysate is determined to be 58.2±5.1 U/mg protein. The method has also been employed to evaluate the inhibitory effect of PKA inhibitors with satisfactory results, and may be expected to be a promising candidate for facile and cost-effective assay of kinase activity and highthroughput inhibitor screening.

The protein kinases superfamily regulates the most frequent post-translational modification through catalyzing the γphosphoryl transfer from adenosine-5’-triphosphate (ATP) to an acceptor, and plays a critical role in many fundamental metabolic and cell-signaling processes.1,2 The aberrant activities of protein kinases may lead to severe human diseases including cancers, Alzheimer’s disease or cardiovascular diseases.2 Furthermore, many inhibitors of protein kinases have been applied to clinical trials and some of them even have been approved for the treatment of cancers.3-5 Therefore, it is of great significance to develop facile and sensitive methods for protein kinases assay and their inhibitor screening. Different detection systems, including radiolabels,6 electrochemical assays,7,8 immunoassays,9,10 mass spectrum,11 colorimetric assays12-14 and so on, have been developed for protein kinases activity assay. Among them, fluorescent methods have attracted much attention due to their inherent advantages such as simple operation, low cost and high throughput.15-38 So far, several fluorescent methods have been proposed to detect protein kinases activity,19-38 among which the small moleculesbased assays are mostly to use metal ions (e.g., Zn2+) to link fluorophores and phosphorylated substrates, and the nanomaterial-based assays are usually to employ the fluorescence quenching effect of gold nanoparticles/graphene oxide or the fluorescence resonance energy transfer (FRET) between organic dyes, quantum dots or upconversion nanoparticles. However, some of these methods suffer from either moderate

sensitivity or the unavailability of nanomaterials. Similarly, researchers have synthesized polymers for determining kinase activity based on concentration quenching39 or FRET,40-42 but it is not easy for biologists or pharmacologists to obtain these tailored polymers. Therefore, there is a need for development of a simple, cost-effective and commercial available platform to detect protein kinases activity and screen inhibitors. Considering that protein kinases can transfer phosphate to specific protein or peptide, our design was started by choosing polymers with multiple phosphates. We envisioned that, after phosphorylation, the electrostatic repulsive force between the phosphates of a polymer and the phosphorylated substrates would alter the spectroscopic signal of the system. On the other hand, natural biopolymer RNA or DNA, capable of combining with cationic peptides,43-46 may serve as a promising candidate to quench the fluorescence of a labeled substrate through concentration quenching. Consequently, we used a commercially available polyuridylic acid (polyU) instead of oligonucleotide in order to form assembly. As a proof of concept, the assay of protein kinase A (PKA), a kind of serine/threonine kinases that phosphorylate the serine residue of the specific substrate (peptide RRASL),47 is demonstrated in this study. The peptide RRASL is labeled with fluorescent 5carboxytetramethylrhodamine (TAMRA), and the electropositive arginines in it facilitate its binding with electronegative polyU through electrostatic attraction, subsequently leading to the generation of nanostructures and causing the fluorescence

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quenching by concentration effect. After reacting with PKA, the peptide will be phosphorylated and then dissociate from polyU, which retrieves the fluorescence (Scheme 1). Scheme 1. The formation of polyU-peptide assembly and its dissociation catalyzed by PKA. ADP: adenosine-5’diphosphate

EXPERIMENTAL SECTION Reagents. Polycytidylic acid potassium salt (polyC), polyU, poly acrylic acid sodium salt (PAA), H-89 dihydrochloride hydrate, H-8 dihydrochloride, bovine serum albumin (BSA), ATP, esterase from porcine liver, ribonuclease A from bovine pancreas, PKA, forskolin and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich. Protein kinase B (Akt1) was purchased from Bio-Techne China Co., Ltd. Dithiothreitol (DTT) was obtained from Alfa Aesar. TAMRAlabeled double(RRASL), triple(RRASL) and RRAPSLRRAPSLRRASL were purchased from GL Biochem (Shanghai, China). Hyaluronic acid (HA, Mw = 3500) was obtained from Yangzhou Zhongfu New Materials Co., Ltd. Staurosporine and imatinib were obtained from J&K Scientific Ltd. All other reagents were of A.R. grade and used as received without further purification. Ultrapure water (over 18 MΩ•cm) from a Milli-Q reference system (Millipore) was used throughout. Apparatus. Transmission electron microscopy (TEM) was measured on a transmission electron microscope HT7700 (Hitachi, Japan). Dynamic light scattering (DLS) and zeta potential analyses were performed on a Zetasizer Nano ZS ZEN3600 (Malvern, United Kingdom). Fluorescence measurements were recorded on a microplate reader (Molecular Devices SpectraMax i3, USA) in 96-well assay plates. General Procedure for PKA Activity Assay. A stock solution of the polyU-peptide assembly was prepared by mixing 5 mL 100 mM HEPES buffer (pH 7.4), 400 µL 100 mM MgCl2, 240 µL 5 mM ATP, 400 µL 50 mM DTT, 200 µL 200 µM TAMRA-labeled triple(RRASL) and 280 µL 250 µg/mL polyU aqueous solution. Then, 163 µL of the assembly stock solution was pipetted to a well of a 96-well assay plate, followed by addition of appropriate volume of PKA solution, and the final volume was adjusted to 250 µL. The final concentrations of different reagents are: 50 mM HEPES, 4 mM MgCl2, 120 µM ATP, 2 mM DTT, 4 µM peptide and 7 µg/mL polyU. After incubated at 37 oC for 1 h, the fluorescence of the reaction solution was recorded with λex/em = 554/585 nm. Detection of PKA Activity in Cell Lysate. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine at 37 oC in a 5% CO2 incubator. Approximate 3×106 HeLa cells were lysed with 0.3 mL lysis buffer (protein extraction kit C500022, Sangon Biotech, Chi-

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na), followed by centrifugation at 12000 rpm for 10 min. The supernate was collected and diluted to 1.8 mL to yield the protein extraction solution (referred to the lysate), in which the total protein concentration was determined to be 0.5 mg/mL by a Lowry protein assay (modified Lowry protein assay kit C504041, Sangon Biotech, China). The activity of PKA in the lysate was assayed following the above procedure. In forskolin and IBMX stimulation experiments, cells were cultured in serum-free medium for 4 h, and then incubated with different concentrations of forskolin and IBMX (in DMSO) for 30 min. In the meantime, the cells treated with equal volume of DMSO were used as unstimulated samples (control). Inhibition Study. The assembly stock solution (163 µL) was mixed with 25 U/mL PKA solution (10 µL) in a 96-well assay plate, followed by addition of appropriate volume of inhibitor solution. The final volume was adjusted to 250 µL in each well. The final concentrations of different reagents are: 50 mM HEPES, 4 mM MgCl2, 120 µM ATP, 2 mM DTT, 4 µM peptide, 7 µg/mL polyU and 1 U/mL PKA. After incubation at 37 oC for 1 h, the fluorescence of the solution was recorded with λex/em = 554/585 nm. The inhibition ratio was calculated using the following equation: inhibition ratio = (∆F0 – ∆Fi)/∆F0 × 100%, where ∆F0 and ∆Fi are the fluorescence enhancement of the reaction system in the absence and presence of inhibitor, respectively. Then, the inhibition ratio (y) was plotted against the varied inhibitor concentration (x), yielding a curve that can be fitted with the following four-parameter equation: a -d y=d+ b x 1+   c where a is the minimum asymptote, b is the Hill’s slope, c is the IC50 value (half maximal inhibitory concentration) to be determined, and d is the maximum asymptote, respectively.

RESULTS AND DISCUSSION Firstly, we examined whether polyU can quench the fluorescence of the TAMRA-labeled peptide through concentration quenching as expected. The double(RRASL) or triple(RRASL) (Scheme S1) rather than RRASL was used to strengthen the affinity. As is seen from Figure 1A, the fluorescence of both the labeled peptides can be efficiently quenched by about 98% by polyU in water. However, in 50 mM HEPES buffer, the fluorescence of double(RRASL) was quenched only by 11%, while the fluorescence quenching (about 93%) of triple(RRASL) was not dramatically influenced. This clearly indicates that the ion strength interferes with the fluorescence quenching of double(RRASL) rather than triple(RRASL), which means that the assembly of triple(RRASL) and polyU is stable enough for application in buffer. In order to insure the low background fluorescence, 7 µg/mL of polyU was used in the subsequent experiments. Under the same polyU concentration, the fluorescence of partially phosphorylated peptide TAMRA-RRAPSLRRAPSLRRASL as a positive control was quenched only by 6% (Figure S1), suggesting that the phosphorylation indeed inhibited the formation of the polyU-peptide assembly. Moreover, to prove the deliberate selection of polyU, other commercially available

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anionic polymers (Scheme S2), including HA, PAA and polyC, were also tested to quench the fluorescence of TAMRA-labeled triple(RRASL). As shown in Figure 1B, only polyC produces effective quenching like polyU. The reason for this may be attributed to the following two aspects: 1, the phosphates bind to the substrate much stronger than carboxylates do; 2, the assembly of multiple pyrimidines causes the inner filter effect of partial fluorescence. B

6

8

4

2 a b b'

0 0

HA

4 PAA

2 PolyC PolyU

0

4 8 PolyU (µg/mL)

0

4 8 Polyanion (µg/mL)

Figure 1. (A) Fluorescence intensity of 4 µM TAMRA-labeled peptides in the presence of varied concentrations (0-9 µg/mL) of polyU. a: double(RRASL) in water; a’: double(RRASL) in 50 mM HEPES (pH 7.4); b: triple(RRASL) in water; b’: triple(RRASL) in 50 mM HEPES (pH 7.4). (B) Fluorescence intensity of 4 µM TAMRA-labeled triple(RRASL) with varied concentrations (0-9 µg/mL) of a polyanion (HA, PAA, polyC or polyU) in 50 mM HEPES (pH 7.4). λex/em = 554/585 nm.

Then, we studied if any nanostructure was formed during this quenching process. The DLS analysis revealed that the average diameter of triple(RRASL) itself is about 240.2±60.0 nm (Figure S2), whereas that of the assembly formed by triple(RRASL) and polyU increases to 532.2±288.4 nm (Figure 2A), suggesting a nanostructure generation. Besides, the TEM analysis showed that the assembly has a random coil like shape with a diameter of about hundreds of nanometers. After PKA was added, the polyU-peptide assembly was disassociated, with a decreased diameter of 214.4±98.78 nm according to the DLS analysis (Figure 2A). During this process, the zeta potential of the assembly went down from -12.4±4.95 mV to 16.3±8.81 mV, according with the expected phosphorylation. Notably, the characteristic fluorescence and absorption spectra of rhodamine can be restored from the quenching system after phosphorylation, accompanied by slight coloration (Figure 2B and Figure S3). A

B 532.2 ± 288.4 nm 8

10 0 20

Fluorescence (×10 )

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214.4 ± 98.78 nm

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Furthermore, as shown in Figure 3, only PKA can ignite the fluorescence of the system; while inorganic salts and other bio-substances, such as NaCl, KCl, CaCl2, CuCl2, glucose, glutathione, esterase and bovine serum albumin (BSA), cause negligible effect on the fluorescence. Notably, Akt1 was also investigated and no fluorescence response was observed. These results suggest the good selectivity toward PKA of the method. PKA

4

8

a'

shows the corresponding color and fluorescent photographs of the system. Reaction condition: polyU (7 µg/mL), peptide (4 µM) in 50 mM HEPES buffer (pH 7.4) with MgCl2 (4 mM), DTT (2 mM), ATP (120 µM) and PKA (4 U/mL) at 37 oC for 1 h.


Fluorescence (× 10 )

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600 640 680 Wavelength (nm)

Figure 2. (A) The DLS and TEM (inset) analyses of the polyUpeptide assembly before (top) and after (bottom) addition of PKA. Scale bar, 1 µm. (B) Fluorescence spectra of the polyU-peptide assembly before (a) and after (b) addition of PKA. The inset

blank, NaCl, KCl, CuCl2, CaCl2, glucose, glutathione, BSA, esterase and Akt1

2

0

600

640 Wavelength (nm)

680

Figure 3. Fluorescence responses of the polyU-peptide assembly to various species: NaCl (5 mM), KCl (5 mM), CaCl2 (500 µM), CuCl2 (100 µM), glucose (5 mM), glutathione (2 mM), esterase (0.25 U/mL), BSA (10 µM), Akt1 (4U/mL) and PKA (3 U/mL).

It is known that polyU as well as RNA can be hydrolyzed by ribonuclease, which is a ubiquitous contaminant. Ribonuclease A was demonstrated to be able to dissociate the present assembly, leading to a fluorescence increase indeed (Figure S4). However, as depicted in Figure S5, the polyU-peptide assembly stays stable for at least 7 days in 50 mM HEPES buffer in vials at 4 oC without obvious fluorescence enhancement, which suggests the good stability of the system under the common laboratory conditions. Next, the influences of reaction time, temperature and pH on PKA activity assay were studied in detail. As shown in Figure S6, the initial fluorescence growth rate is fast and dependent on the amount of PKA used. However, a relatively stable and strong fluorescence can be observed in about 60 min. Figure S7 reveals that the fluorescence of the assembly itself is almost unchanged at the temperature ranging from 25 to 42 oC, but the fluorescence signal of the system with PKA reaches the largest increase at 37 oC, indicating that PKA works well at the normal body temperature. As depicted in Figure S8, the fluorescence of the system without PKA is relatively low in pH 7.4 media, and both weak acid and alkaline conditions could elevate the background signal, probably due to the slight dissociation of the assembly by extra charges introduced by acid or base. After adding PKA, reaction at about pH 7.4 produces a larger fluorescence enhancement. Hence, the optimized condition of the reaction at 37 oC and pH 7.4 for 60 min was used for the subsequent PKA activity assay and inhibitor screening. Figure 4A demonstrates the fluorescence response of the system to PKA at varied concentrations. The fluorescence intensity increases as PKA rises from 0.1 to 4.4 U/mL, with a linear equation of ∆F = 1.23×108 [PKA] − 5.97×106 (R2 =

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Figure 4. (A) Fluorescence spectra of the polyU-peptide assembly in the presence of varied concentrations of PKA (0, 0.1, 0.2, 0.4, 0.8, 1.0, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0 and 4.4 U/mL). (B) Linearity between the fluorescence intensity change and the concentration of PKA (∆F = F − F0, where F and F0 are the fluorescence intensities after and before addition of PKA, respectively). λex/em = 554/585 nm.

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The applicability of our method was demonstrated for PKA analysis in complex cell lysate. Addition of 10 µL HeLa cell lysate to the assembly solution produces significant fluorescence enhancement (Figure 5D), from which the activity of PKA is determined to be 58.2±5.1 U/mg protein in HeLa cells, which is in agreement with that obtained by electrophoresis.49 As positive controls, forskolin (activating adenylyl cyclase) and IBMX (an effective inhibitor of phosphodiesterase)29, which are able to stimulate PKA activity in cells, were utilized to treat HeLa cells, and the lysate of the as-treated HeLa cells was also analyzed in parallel. As shown in Figure 5F or 5G, the fluorescence from the treated cells is much larger than that from the untreated ones. Notably, all of these fluorescence increases can be suppressed by staurosporine (an effective inhibitor of PKA50), as depicted in Figure 5E or 5H. Collectively, these phenomena suggest that our method can determine the PKA activity and its variation in complex biological samples. 2

in Figure 6, the inhibition efficiencies for PKA of the four inhibitors go up with concentrations. Sigmoidal fitting was applied to plot the inhibition curve, which reveals that the order of inhibitory effect of the four inhibitors is: staurosporine>H-89>H-8>imatinib, and the IC50 values for the former three are 9.02±1.07 nM, 142.9±13.1 nM and 4.54±0.41 µM, respectively, which are consistent with the reported results.50-52 As for imatinib, which is not a specific inhibitor for PKA, the IC50 value is too high to be determined reliably due to its poor inhibitory effect for PKA. These data indicate that the polyUpeptide assembly is also suitable for the screening of PKA inhibitors. 100 Inhibition Ratio (%)

0.993) in the range of 0.1 - 3.2 U/mL PKA. The detection limit (3S/N, n = 11)48 was determined to be 0.05 U/mL.


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S

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I

0 1E-4 1E-3 0.01 0.1 1 Inhibitor (µM)

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Figure 6. Inhibition curves of different inhibitors: staurosporine (S), H-89, H-8 and imatinib (I).

CONCLUSION We have developed a facile method for PKA activity assay based on the assembly of polyU and the TAMRA labeled triple(RRASL). The fluorescence of the assembly is quenched due to concentration effect, and can be selectively retrieved by the phosphorylation of PKA. The method is sensitive, and rather simple (Table S1), and can be used to determine the PKA activity in complex biological samples and screen inhibitors of PKA. It is worth mentioning that, in our strategy, polyU, as well as polyC, is commercially available and can be used directly as a quencher of a cationic fluorescent peptide, which encourages us to explore the versatility of polynucleotides, especially more stable and cheaper DNAs,45,46 as quencher for developing facile analytical platforms of other substances.

ASSOCIATED CONTENT Supporting Information

1

The Supporting Information is available free of charge on the ACS Publications website. 0

A

B

C D

E

F

G H

Figure 5. Detection of PKA activity in HeLa cell lysate. A: blank; B: lysis buffer; C: lysis buffer + 25 µM forskolin/50 µM IBMX; D: cell lysate; E: cell lysate + staurosporine; F: cell lysate + 10 µM forskolin/20 µM IBMX; G: cell lysate + 25 µM forskolin/50 µM IBMX; H: system G + staurosporine.

Further, we examined whether the method can be used to screen inhibitors of PKA. In this experiment, three known inhibitors (staurosporine, H-89, H-8; Scheme S3) for PKA and one specific inhibitor (imatinib) for Bcr-Abl tyrosine kinase were studied and their IC50 values were determined. As shown

The structures of peptides, polyanions and inhibitors, comparison of quenching ability between peptides and phosphorylated peptides, DLS plots, absorption spectra, the stability study of polyUpeptide assembly, the effect of incubation time, temperature and pH on the PKA assay and the comparison of different assays for PKA activity (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +86-10-62554673

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Notes

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The authors declare no competing financial interest.

ACKNOWLEDGMENT We are grateful for the financial support from the NSF of China (Nos. 21675159, 21435007, 21535009, and 21621062), the 973 Program (Nos. 2015CB932001 and 2015CB856301), the Chinese Academy of Science (XDB14030102), and Youth Innovation Promotion Association of CAS (2016027).

REFERENCES (1) Shiloh, Y.; Ziv, Y. Nat. Rev. Mol. Cell Biol. 2013, 14, 197-210. (2) Gonzalez-Vera, J. A. Chem. Soc. Rev. 2012, 41, 1652-1664. (3) Dar, A. C.; Shokat, K. M. Annu. Rev. Biochem. 2011, 80, 769795. (4) Liu, X.; Winey, M. Annu. Rev. Biochem. 2012, 81, 561-585. (5) Wang, L. J.; Yang, Y.; Zhang, C. Y. Anal. Chem. 2015, 87, 4696-4703. (6) Hutti, J. E.; Jarrell, E. T.; Chang, J. D.; Abbott, D. W.; Storz, P.; Toker, A.; Cantley, L. C.; Turk, B. E. Nat. Methods. 2004, 1, 2729. (7) Shin, I. S.; Chand, R.; Lee, S. W.; Rhee, H. W.; Kim, Y. S.; Hong, J. I. Anal. Chem. 2014, 86, 10992-10995. (8) Zhao, H. F.; Liang, R. P.; Wang, J. W.; Qiu, J. D. Chem. Commun. 2015, 51, 12669-12672. (9) Farley, K.; Mett, H.; McGlynn, E.; Murray, B.; Lydon, N. B. Anal. Biochem. 1992, 203, 151-157. (10) Schraag, B.; Staal, G. E. J.; Adriaansenslot, S. S.; Salden, M.; Rijksen, G. Anal. Biochem. 1993, 211, 233-239. (11) Kim, Y. P.; Oh, E.; Oh, Y. H.; Moon, D. W.; Lee, T. G.; Kim, H. S. Angew. Chem., Int. Ed. 2007, 46, 6816-6819. (12) Wang, Z. X.; Levy, R.; Fernig, D. G.; Brust, M. J. Am. Chem. Soc. 2006, 128, 2214-2215. (13) Wei, H.; Chen, C.; Han, B.; Wang, E. Anal. Chem. 2008, 80, 7051-7055. (14) Zhou, J.; Xu, X.; Liu, X.; Li, H.; Nie, Z.; Qing, M.; Huang, Y.; Yao, S. Biosens. Bioelectron. 2014, 53, 295-300. (15) Li, X. H.; Gao, X. H.; Shi, W.; Ma, H. M. Chem. Rev. 2014, 114, 590-659. (16) Li, L. H.; Shi, W.; Wang, Z.; Gong, Q. Y.; Ma, H. M. Anal. Chem. 2015, 87, 8353-8359. (17) Wu, X. F.; Li, L. H.; Shi, W.; Gong, Q. y.; Ma, H. M. Angew. Chem., Int. Ed. 2016, 55, 14728-14732. (18) He, X. Y.; Xu, Y. H.; Shi, W.; Ma, H. M. Anal. Chem. 2017, 89, 3217-3221. (19) Akita, S.; Umezawa, N.; Higuchi, T. Org. Lett. 2005, 7, 55655568. (20) Agnes, R. S.; Jernigan, F.; Shell, J. R.; Sharma, V.; Lawrence, D. S. J. Am. Chem. Soc. 2010, 132, 6075-6080. (21) Chen, C. A.; Yeh, R. H.; Lawrence, D. S. J. Am. Chem. Soc. 2002, 124, 3840-3841. (22) Shults, M. D.; Imperiali, B. J. Am. Chem. Soc. 2003, 125, 14248-14249. (23) Rhee, H. W.; Lee, S. H.; Shin, I. S.; Choi, S. J.; Park, H. H.; Han, K.; Park, T. H.; Hong, J. I. Angew. Chem., Int. Ed. 2010, 49, 4919-4923. (24) Kikuchi, K.; Hashimoto, S.; Mizukami, S.; Nagano, T. Org. Lett. 2009, 11, 2732-2735. (25) Lu, G. Y.; Tan, P. L.; Lei, C. Y.; Nie, Z.; Huang, Y.; Yao, S. Z. Talanta 2014, 128, 360-365. (26) Zhou, J.; Xu, X. H.; Liu, W.; Liu, X.; Nie, Z.; Qing, M.; Nie, L. H.; Yao, S. Z. Anal. Chem. 2013, 85, 5746-5754. (27) Liu, C. H.; Chang, L. J.; Wang, H. H.; Bai, J.; Ren, W.; Li, Z. P. Anal. Chem. 2014, 86, 6095-6102. (28) Moon, J. H.; MacLean, P.; McDaniel, W.; Hancock, L. F. Chem. Commun. 2007, 4910-4912.

(29) Bai, J.; Zhao, Y. J.; Wang, Z. B.; Liu, C. H.; Wang, Y. C.; Li, Z. P. Anal. Chem. 2013, 85, 4813-4821. (30) Tan, P. L.; Lei, C. Y.; Liu, X.; Qing, M.; Nie, Z.; Guo, M. L.; Huang, Y.; Yao, S. Z. Anal. Chim. Acta. 2013, 780, 89-94. (31) Yang, W. X.; Wang, Y. C.; Chang, L. J.; Liu, C. H.; Bai, J.; Li, Z. P. RSC Adv. 2014, 4, 14546-14549. (32) Ren, W.; Liu, C. H.; Lian, S.; Li, Z. P. Anal. Chem. 2013, 85, 10956-10961. (33) Zhang, X. B.; Liu, C. H.; Wang, H. H.; Wang, H.; Li, Z. P. Angew. Chem., Int. Ed. 2015, 54, 15186-15190. (34) Xu, X. H.; Liu, X.; Nie, Z.; Pan, Y. L.; Guo, M. L.; Yao, S. Z. Anal. Chem. 2011, 83, 52-59. (35) Shen, C. C.; Xia, X. D.; Hu, S. Q.; Yang, M. H.; Wang, J. X. Anal. Chem. 2015, 87, 693-698. (36) Song, W.; Liang, R. P.; Wang, Y.; Zhang, L.; Qiu, J. D. Chem. Commun. 2015, 51, 10006-10009. (37) Song, W.; Liang, R. P.; Wang, Y.; Zhang, L.; Qiu, J. D. Sens. Actuators, B: Chem. 2016, 226, 144-150. (38) Ma, F.; Li, Y.; Tang, B.; Zhang, C.Y. Acc. Chem. Res. 2016, 49, 1722-1730. (39) Koga, H.; Toita, R.; Mori, T.; Tomiyama, T.; Kang, J. H.; Niidome, T.; Katayama, Y. Bioconjugate Chem. 2011, 22, 1526-1534. (40) Bai, J.; Liu, C. H.; Yang, T.; Wang, F. F.; Li, Z. P. Chem. Commun. 2013, 49, 3887-3889. (41) Toita, R.; Mori, T.; Naritomi, Y.; Kang, J. H.; Shiosaki, S.; Niidome, T.; Katayama, Y. Anal. Biochem. 2012, 424, 130-136. (42) Nobori, T.; Shiosaki, S.; Mori, T.; Toita, R.; Kim, C. W.; Nakamura, Y.; Kishimura, A.; Niidome, T.; Katayama, Y. Bioconjugate Chem. 2014, 25, 869-872. (43) Kamat, N. P.; Tobe, S.; Hill, I. T.; Szostak, J. W. Angew. Chem., Int. Ed. 2015, 54, 11735-11739. (44) Aumiller, W. M., Jr.; Keating, C. D. Nat. Chem. 2016, 8, 129137. (45) Han, Y. T.; Li, H.; Hu, Y. F.; Li, P.; Wang, H. X.; Nie, Z.; Yao, S. Z. Anal. Chem. 2015, 87, 9179-9185. (46) Wang, H. X.; Li, Y.; Zhao, K. L.; Chen, S. Y.; Wang, Q.; Lin, B.; Nie, Z.; Yao, S. Z. Biosens. Bioelectron. 2017, 91, 400-407. (47) Kemp, B. E. J. Biol. Chem. 1980, 255, 2914-2918. (48) Wang, Z.; Li, X. H.; Song, Y. C.; Li, L. H.; Shi, W.; Ma, H. M. Anal. Chem. 2015, 87, 5816-5823. (49) Humphries, K. M.; Pennypacker, J. K.; Taylor, S. S. J. Biol. Chem. 2007, 282, 22072-22079. (50) Li, Q.; Li, T. M.; Zhu, G. D.; Gong, J. C.; Claibone, A.; Dalton, C.; Luo, Y.; Johnson, E. F.; Shi, Y.; Liu, X. S.; Klinghofer, V.; Bauch, J. L.; Marsh, K. C.; Bouska, J. J.; Arries, S.; De Jong, R.; Oltersdorf, T.; Stoll, V. S.; Jakob, C. G.; Rosenberg, S. H.; Giranda, V. L. Bioorg. Med. Chem. Lett. 2006, 16, 1679-1685. (51) McHardy, T.; Caldwell, J. J.; Cheung, K. M.; Hunter, L. J.; Taylor, K.; Rowlands, M.; Ruddle, R.; Henley, A.; Brandon, A. d. H.; Valenti, M.; Davies, T. G.; Fazal, L.; Seavers, L.; Raynaud, F. I.; Eccles, S. A.; Aherne, G. W.; Garrett, M. D.; Collins, I. J. Med. Chem. 2010, 53, 2239-2249. (52) Lavogina, D.; Lust, M.; Viil, I.; Koenig, N.; Raidaru, G.; Rogozina, J.; Enkvist, E.; Uri, A.; Bossemeyer, D. J. Med. Chem. 2009, 52, 308-321.

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