Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe

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Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform Mingxuan Gao, Jingjing Guo, Yanling Song, Zhi Zhu, and Chaoyong James Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14185 • Publication Date (Web): 13 Oct 2017 Downloaded from http://pubs.acs.org on October 15, 2017

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ACS Applied Materials & Interfaces

Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform Mingxuan Gao,a Jingjing Guo,a Yanling Song,*a,b Zhi Zhu,*a Chaoyong James Yang*a a. MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Collaborative Innovation Centre of Chemistry for Energy Materials, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Chronic Liver Disease and Hepatocellular Carcinoma of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China. b. The Key Lab of Analysis and Detection Technology for Food Safety of MOE, State Key Laboratory of Photocatalysis on Energy and Environment, College of Biological Science and Engineering, Fuzhou University, Fuzhou 350116, China.

* To whom correspondence should be addressed. Tel: (+86) 592-218-7601; Fax: (+86) 592-2189959. E-mail: [email protected]; or Tel: (+86) 591-22860973; E-mail: [email protected]

KEYWORDS: Detection, T4 PNK, aptamer, DNA, fluorescence

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ABSTRACT: As a vital enzyme in DNA phosphorylation and restoration, T4 polynuceotide kinase (T4 PNK) has aroused great interest in recent years. Therefore, numerous strategies have been established for highly sensitive detection of T4 PNK based on diverse signal amplification techniques. However, they often need sophisticated design, a variety of auxiliary reagents and enzymes, or cumbersome manipulations. We have designed a new kind of allosteric aptamer probe consisting of streptavidin aptamer (SA aptamer) and the complementary DNA (cDNA) for simple detection of T4 PNK without signal amplification and with minimized interference in complex biological samples. When the 5’-terminus of the cDNA is phosphorylated by T4 PNK, the cDNA is degraded by Lambda exonuclease (λ exo) to release the FAM-labelled SA aptamer, which subsequently binds to streptavidin beads (SA beads). The enhancement of the fluorescence signal on SA beads can be detected precisely and easily by a microscope or flow cytometer. Our method performs well in complex biological samples as a result of the enrichment of the signaling molecules on beads, as well as simple manipulations to discard the background interference and non-binding molecules. Without signal amplification techniques, our allosteric aptamer probe method not only avoids complicated manipulations but also decreases the time required. With the advantages of ease of operation, reliability and robustness for T4 PNK detection in buffer as well as real biological samples, the allosteric aptamer probe has great potential for clinical diagnostics, inhibitor screening and drug discovery.


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INTRODUCTION DNA phosphorylation, which occurs in nearly every cell, is of great consequence in the rehabilitation of DNA damage. T4 polynucleotide kinase (T4 PNK) is one kind of functional enzyme specifically for the phosphorylation at the 5’-OH via the transfer of the γ-phosphate from ATP to DNA. This process plays a crucial part in DNA recombination and replication, as well as restoration.1-6 Many researches have demonstrated that aberrant activity of T4 PNK, which induces abnormal phosphorylation, is related to some serious human diseases such as RothmundThomson Syndrome, Werner Syndrome and Bloom’s Syndrome.7, 8 Accordingly, it is of great importance to establish a highly sensitive detection strategy for T4 PNK, for use in inhibitor screening, clinical diagnostics and drug discovery. Polyacrylamide gel electrophoresis (PAGE) associated with radical isotope P-labelling is the conventional standard method for T4 PNK determination.9-11 However, there are several inevitable drawbacks of this method, such as exposure of the operator to radiation, as well as complicated and time-consuming manipulations. In order to solve these problems, numerous effective strategies have been developed, including colorimetric methods,12, 13 electrochemical methods,14-16,

51

nanoparticle-based methods17-19,

45

and fluorescence methods.6,

20-32, 46-48, 52

Among them, a fluorescence-mediated strategy has been widely applied in T4 PNK detection due to its high sensitivity, simple manipulations and easy signal readout. For instance, Song et al. initially developed a single-labelled DNA molecular beacon for determination of T4 PNK associated with Lambda exonuclease (λ exo) digestion.20 Lin et al. used graphene oxide as the effective quencher and carrier to establish a fluorescence-based detection method.21 To further improve the sensitivity, several signal amplification techniques have been applied, including hybridization chain reaction (HCR)22, nicking enzyme signal amplification (NESA)31, strand


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displacement reaction (SDR)23, hyperbranched rolling circle amplification (HRCA)46 and multiple amplification47. However, the fluorescence methods still exhibit several limitations, including complicated probe design, the need for different kinds of auxiliary enzymes and reagents, and the apparent loss of sensitivity in complex biological systems. For instance, Zhang et al. only used DMEM cell medium to simulate the detection environment of a complex solution.22 Cheng et al. found that the lowest detectable concentration increased one order of magnitude in 50% HeLa cell extracts.23 As a consequence, a robust yet reliable fluorescence detection method with sensitivity and selectivity in complex biological samples is still needed. Previously, we have established allosteric molecular beacon (aMB) platforms for highly sensitive detection of different types of analytes, including small molecules, proteins and nucleic acids, both in buffer and in real complex biological samples.33-35 In short, aMB is a singlestranded DNA which contains three parts: a molecular recognition region, a fluorophore-labelled streptavidin (SA) aptamer36, 37 for signal output, and a complementary DNA (cDNA) sequence of SA aptamer. When the target is absent, cDNA hybridizes with SA aptamer to form a hairpin structure which restricts the binding of SA aptamer to the SA beads. On the contrary, when target exists, it reacts with the target-recognition region, which changes the conformation of aMB and liberates SA aptamer to bind to SA beads, resulting in fluorescence intensity increase of SA beads. The enhancement of the fluorescent signal on SA beads can be detected precisely and rapidly by a microscope or flow cytometer. Compared with other aptamer-based detection method, aMB possesses more versatility, for it doesn’t need the aptamer of the target molecule itself. Simply, only specific target-recognition region and common SA aptamer are necessary.4950


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By harnessing the merits of aMB, we applied the aMB-based strategy for quantitative detection of T4 PNK. Furthermore, we designed and optimized a particular double-stranded aMB named allosteric aptamer probe (AAP) for this system. The AAP is composed simply of a fluorophore-labelled SA aptamer and its full length of cDNA with 5’-OH. When the 5’-terminus of the cDNA is phosphorylated by T4 PNK, the cDNA will be degraded by Lambda exonuclease (λ exo) and release the FAM-labelled SA aptamer, which will subsequently bind to streptavidin beads (SA beads). Our method performs well in complex biological samples as a result of the enrichment of the signalling molecules on beads, as well as simple manipulations to discard the background interference and non-binding molecules. Because it does not require signal amplification techniques, the AAP avoids complicated manipulations but saves time. With the advantages of ease of operation, reliability and robustness for T4 PNK detection in buffer as well as in real biological samples, the AAP has great potential for clinical diagnostics, inhibitor screening and drug discovery. EXPERIMENTAL SECTION Chemicals and reagents. T4 PNK, λ exo and T4 DNA ligase were purchased from New England Biolabs (NEB, UK). Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and lysozyme were purchased from Sangon Biotech Co. (Shanghai, China). Alkaline phosphatase (ALP) was purchased from Thermo Fisher Scientific Inc (Waltham, MA, USA). C-reactive protein (CRP) was purchased from R&D Systems (Minneapolis, MN, USA). Catalase was purchased from Worthington Biochemistry Co. (Lakewood, NJ, USA). Trypsin was purchased from GIBCO (Waltham, MA, USA). Thrombin was purchased from Haematologic Technologies (Essex Junction, VT, USA). Albumin from bovine serum (BSA) was purchased from Tagene Biotechnology Co. (Xiamen, China). Streptavidin beads were purchased from GE Healthcare


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(Chicago, IL, USA). The oligonucleotides used in this assay were all synthesized by Sangon Biotech Co. (Shanghai, China), and the sequences are listed in Table. 1. The underlined nucleotides are extended sequences in the optimization experiment. Ultrapure water (18.2 MΩ cm-1) was obtained from an OMNI water purification system of Research Scientific Instruments Co. (Xiamen, China). All other chemicals were of analytical grade. Table 1. DNA sequences used. Name

Sequence

SA-0

5'-FAM-ATT GAC CGC TGT GTG ACG CAA CAC TCA AT-3'

cDNA-0

5'-ATT GAG TGT TGC GTC ACA CAG CGG TCA AT-3'

SA-3

5'-FAM-CGC ATT GAC CGC TGT GTG ACG CAA CAC TCA AT-3'

cDNA-3

5'-ATT GAG TGT TGC GTC ACA CAG CGG TCA ATG CG-3'

SA-6

5'-FAM-CTC CGC ATT GAC CGC TGT GTG ACG CAA CAC TCA AT-3'

cDNA-6

5'-ATT GAG TGT TGC GTC ACA CAG CGG TCA ATG CGG AG-3'

SA-9

5'-FAM-CCC CTC CGC ATT GAC CGC TGT GTG ACG CAA CAC TCA AT-3'

cDNA-9

5'-ATT GAG TGT TGC GTC ACA CAG CGG TCA ATG CGG AGG GG-3'

Synthesis and optimization of AAP. First, to prepare the double-stranded AAP, 10 µM SA aptamer and 10 µM corresponding cDNA in reaction buffer (70 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT, pH=8.0) were heated to 95ºC for 5 min and cooled to room temperature gradually for 1 h. Then 200 nM AAP, 1 mM ATP, 80 U/mL λ exo and 1 U/mL T4 PNK (without T4 PNK as a control group) were mixed in 100 µL buffer and incubated at 37ºC for 3 h, followed by heating to 75ºC for 10 min to deactivate the enzymes and cooling to the room temperature. Afterwards, 3 µL SA beads (about 4×104 beads) were added and incubated at room temperature for 5 min on a rotator. Finally, the beads were washed with buffer 3 times, and the fluorescence intensity of the beads was quantitatively determined by flow cytometer (FACSVerse, BD, USA).


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Gel electrophoresis. The pivotal reaction including the phosphorylation and the digestion was validated by PAGE. Ten µL 500 nM AAP solution was digested by 100 U/mL T4 PNK and 80 U/mL λ exo, and was then mixed with 2 µL loading buffer as a sample for 12.5% native PAGE. The electrophoresis was performed at a constant voltage of 120 V for 55 min in 1 × TBE buffer (100 mM Tris-HCl, 83 mM boric acid, 1 mM EDTA, pH=8.0), and after staining and decolorization, the image was photographed with a Canon camera. Preparation of cell lysates. To investigate the applicability of this method in biological samples, we used HeLa cell lysates as the reaction system. HeLa cells were initially cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum at 37ºC in a cell incubator under an atmosphere of 5% CO2. After the incubation, HeLa cells were digested with trypsin and resuspended in DMEM, and subsequently counted and diluted. Then the suspension was centrifuged at 1000 rpm for 3 min. The supernatant was discarded and the cells were washed 3 times with buffer and resuspended in 1 mL reaction buffer. Afterwards, HeLa cells were sonicated for 10 s at 30 s intervals for 5 cycles. To remove the cell fragments, the suspension was centrifuged at 14000 rpm for 30 min at 4ºC. Ultimately, the supernatant was used in the subsequent experiments. Detection performance of AAP platform. To perform the quantitative detection of T4 PNK both in reaction buffer and complex biological samples, a series of concentrations of T4 PNK with 200 nM AAP, 1 mM ATP, and 80 U/mL λ exo were added to 100 µL reaction buffer or HeLa cell lysates and treated as described above in Synthesis and Optimization of AAP. Furthermore, in order to test the specificity of this method, lysozyme, thrombin, catalase, BSA, CRP, T4 DNA ligase and inactive T4 PNK were added respectively as substitutions of T4 PNK. The concentrations of proteins including BSA, CRP, catalase and thrombin were 10 nM. And the


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concentrations of enzymes including T4 ligase, lysozyme and deactivated T4 PNK were 10 U/mL, which were ten times that of T4 PNK. The samples without analytes were control groups. T4 PNK inhibition study. Ammonium sulfate and ADP, which are commonly used as model inhibitors for T4 PNK, 21, 23, 38 were chosen to perform the inhibition study. The concentration of the inhibitors was 0-11 mM for ammonium sulfate and 0-60 mM for ADP. The experiment steps were same as stated above with 1 U/mL T4 PNK in buffer. RESULTS AND DISCUSSION Scheme 1. Schematic illustration of AAP-based T4 PNK detection method

Working principle. For highly sensitive detection of T4 PNK both in reaction buffer and biological samples, we designed AAP to mimic the allosteric effect in nature. The alteration of the AAP conformation controls the binding affinity of the SA aptamer. As a consequence, the presence of T4 PNK induces alteration of the AAP conformation to increase the binding affinity of the SA aptamer. This alteration can be read out easily after transforming to a fluorescence signal. As shown in Scheme 1, in the presence of T4 PNK, the 5’-OH of cDNA is phosphorylated and is able to be recognized by λ exo, resulting in degradation of the cDNA. Then the released SA aptamer binds to SA beads and increases the fluorescence intensity of the


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beads, which can be quantitatively detected by microscope or flow cytometer. However, in the absence of T4 PNK, the binding affinity of SA aptamer towards SA is blocked by cDNA. Thus, degradation of AAP is not activated and no signal is produced. Moreover, the intact AAPs not bound to the SA beads are easily isolated by simple washing and filtration after only 5 min incubation, thereby decreasing the nonspecific adsorption-induced background sharply and decreasing time-consumption for signal readout. In this system, λ exo is more inclined to degrade one of the dsDNAs having a 5’PO4 group.8, 39 If a single-stranded stem-loop probe is used, the degradation can be stopped in advance due to the stem structure is destroyed by the degradation itself. Therefore, the double-stranded AAP, which has a more stable conformation than previous single-stranded aMBs, is more compatible during the degradation reaction. Synthesis and optimization of AAP. Optimization experiments were essential due to the competitive relationship between the cDNA and SA beads towards SA aptamer. A stronger affinity to SA beads would lead to a high background signal. The length of the probe would have considerable influence on the signal-to-background (S/B) ratio. In order to acquire the best S/B ratio, we designed SA-3, SA-6 and SA-9 (Table 1), which have longer sequences than the original SA aptamer (SA-0), to form a more stable double-stranded structure. After hybridization with their corresponding full length cDNAs, the aptamers changed into double-stranded forms, which were applied to detect 1 U/mL T4 PNK, respectively. As shown in Fig 1, SA-0 had a low S/B ratio mainly due to its relatively unstable double-stranded conformation with its cDNA, which induces high background. With the sequence extended, the background gradually decreased. However, the background of SA-9 increased to some extent. For this, we found similar result in the previous work of Shangguan et al. that the extension of the stem region of SA aptamer could slightly increase its binding affinity towards streptavidin (St-2-A, which was a


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derivative from St-2-1, showing higher binding affinity than its original sequence (Table S2)).37 They also explained that this cannot increase the binding affinity of SA aptamer much, and thus we can only see slightly increasing background between SA-6 and SA-9. On the other hand, longer sequence can also be an important reason for the low S/B ratio of SA-9. In comparison, the signal change upon addition of T4 PNK was not the decisive factor due to its lesser contribution to S/B ratio (less than two times between the maximum and the minimum). Therefore, SA-6 with lowest background had the best S/B ratio of 193, one to two orders of magnitude higher than S/Bs of other fluorescence methods.5, 26-28 As a consequence, our method exhibits substantial capability for a low limit of detection, and SA-6 was chosen to be used in subsequent experiments.

Figure 1. Optimization of aptamer sequence. (A) Signal and background fluorescence intensity using different AAPs. Red line shows the fluorescence intensity of the SA beads with 1 U/mL T4 PNK. Blue line shows the fluorescence intensity of the SA beads without T4 PNK as background signal. (B) S/B ratio of different AAPs. SA-6 shows the highest S/B ratio mainly due to its lowest background signal.


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Feasibility test of AAP platform. In order to test the feasibility of the AAP system, we first monitored the enzyme digestion reaction via gel electrophoresis. As shown in Fig 2A, Lane 3 indicates good hybridization of SA-6 and cDNA-6 to form the double-stranded AAP. Lane 5 with both T4 PNK and λ exo have significantly decreased AAP band intensity as well as recovered SA-6 band. In the absence of λ exo (lane 4) or T4 PNK (lane 6) or in the presence of inactive T4 PNK (lane 7), the AAP band remained intact. Therefore, the gel electrophoresis experiment validates the phosphorylation of the cDNA on 5’-terminus with T4 PNK and specific degradation of the phosphorylated cDNA by λ exo.

Figure 2. Feasibility validation via gel electrophoresis and fluorescence microscope imaging. (A) 12.5% native PAGE. Lane 1, SA-6; Lane 2, cDNA-6; Lane 3, SA-6 and cDNA-6; Lane 4, SA-6, cDNA-6, and T4 PNK; Lane 5, SA-6, cDNA-6, T4 PNK, and λ exo; Lane 6, SA-6, cDNA-6, and λ exo; Lane 7, SA-6, cDNA-6, λ exo, and inactive T4 PNK. (B) Fluorescence microscope images of SA beads with and without T4 PNK. The left two images are taken in bright field and the right ones are taken in fluorescent field.


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Fluorescence microscope imaging was used to verify the aptamer and bead-based signal output. In Fig 2B, the SA beads showed high fluorescence intensity in the presence of 1 U/mL T4 PNK while aphotic without T4 PNK. The results verified that without T4 PNK the fluorophore-labelled SA aptamer was inactive in the AAP duplex, while with T4 PNK the degradation of phosphorylated cDNA liberated SA aptamer to bind with SA beads and generate highly fluorescent beads. Optimization of reaction conditions. As ATP is a prerequisite of the phosphorylation, we further optimized the concentration of ATP. In Fig 3A, the S/B ratio increased accompanying the gradual increase of ATP concentration, and it reached a maximum at the concentration of 1 mM. Further increasing the ATP concentration to 5 mM and 10 mM resulted in dramatically decreased S/B ratio, probably due to competitive binding between AAP and ATP towards T4 PNK when excess ATP was present. As a result, 1 mM was chosen for further experimentation. Similarly, the concentration of λ exo is also important. Excessive λ exo would induce nonspecific digestion and waste of chemicals, while insufficient λ exo would result in low digestion efficiency, both of which would decrease the S/B ratio. As shown in Fig 3B, 80 U/mL was the optimal concentration which was chosen for further experiments.

Figure 3. Optimization of the concentration of (A) ATP and (B) λ exo to obtain best S/B ratio. Error bars are standard deviations (S/N=3).


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Quantitative detection of T4 PNK. With the optimized conditions, we detected a series of concentrations of T4 PNK via AAP by flow cytometry. As shown in Fig 4A, the fluorescence intensity of SA beads increased gradually while the concentration of T4 PNK increased from 0 to 1 U/mL. With higher concentration of T4 PNK, more AAP was phosphorylated and digested, thereby inducing higher fluorescence intensity. As shown in Fig 4C, by plotting the fluorescence intensity (average of three samples) versus the T4 PNK concentration, the result revealed linear response (R2=0.997) and a detection limit of 0.01 U/mL. Moreover, verification via fluorescence microscope also indicated that the alteration of the fluorescence intensity on SA beads can be monitored simply by visual assessment. As shown in Fig 4E, the fluorescence intensity on SA beads increased intuitively as the concentration of T4 PNK increased.

Figure 4. Flow cytometry data of quantitative detection of T4 PNK in (A) buffer and (B) HeLa cell lysates with concentration ranges of 0-1 U/mL in buffer and 0-10 U/mL in cell lysates. The working curve and linear range for quantitative detection of T4 PNK in (C) buffer and (D) HeLa


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cell lysates. The error bars were obtained from three parallel tests. (E) Signal readout of AAP system in buffer via fluorescence microscope. Scale bars are 100 µm. Relying on the working principle of AAP, signaling molecules are enriched on SA beads, while other non-target molecules can be easily discarded via filtration, which endows the AAP based detection method with strong anti-interference capability. Therefore, the AAP based detection assay was further performed in HeLa cell lysates. As shown in Fig 4B and 4D, the fluorescence intensity still increased accompanying the increase of T4 PNK concentration. Furthermore, the method maintained a good detection limit of 0.05 U/mL in HeLa cell lysates as well as a good linear response (R2=0.962) (from 0.05 U/mL to 0.5 U/mL) compared with other detection methods,22,

23, 40

further indicating that AAP possesses great potential for clinical

application. Specificity of the T4 PNK detection assay. In order to investigate the selectivity of the method, several control proteins including CRP, BSA, thrombin, catalase, lysozyme, T4 DNA ligase and heat-inactive T4 PNK were chosen as substitutes for T4 PNK. The concentrations of proteins including BSA, CRP, catalase and thrombin were 10 nM. And the concentrations of enzymes including T4 ligase, lysozyme and inactive T4 PNK were 10 U/mL, which is ten times that of T4 PNK. As shown in Fig 5A and 5B, only T4 PNK produced a significant increase of fluorescence intensity, which was two orders of magnitude higher than that of other common proteins or enzymes. This result indicates the excellent specificity of the AAP-based detection method.


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Inhibitor screening of T4 PNK. As aberrant activity of T4 PNK induces some types of serious human diseases,

7, 8

the evaluation of inhibitors against T4 PNK will have great

significance in drug discovery and clinical therapeutics. Herein, we selected two inhibitors reported in previous work, ADP and ammonium sulfate, as models for inhibitor screening.21, 23, 32, 38

ADP inhibits the phosphorylation, probably by hindering the phosphate transfer from ATP

to DNA; while ammonium sulfate influences the phosphorylation by salt effects which possibly change the conformation of T4 PNK into an inactive state. As shown in Fig 5C and 5D, with increasing concentrations of ADP and ammonium sulfate, the fluorescence intensity decreased and reached a plateau when the inhibitor concentration reached a certain level. The IC50 (the concentration of inhibitor when half of the enzyme activity is inhibited) of ADP and ammonium sulfate were 2.0 mM and 7.5 mM, respectively, which were similar to those in previously reported work.23 The result indicates that the inhibition ability of ADP is higher than that of ammonium sulfate, and also proved that the AAP-based detection method can be used as a platform for inhibitor screening.


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Figure 5. (A) Specificity testing of the T4 PNK assay by flow cytometry with control proteins of CRP, BSA, thrombin, catalase, lysozyme, T4 DNA ligase and heat-inactive T4 PNK. (B) The statistics of specificity experiment. Inhibition study of T4 PNK with (C) ADP and (D) Ammonium sulfate. Error bars in figures are standard deviations (S/N=3). CONCLUSIONS In conclusion, we have designed an allosteric aptamer probe (AAP) for T4 PNK detection with high sensitivity and selectivity. Only a single-fluorophore-labelled DNA probe, λ exo and SA beads are needed, without any other intricate signal amplification apparatus or reagents. Moreover, compared with other detection methods (Table S1)

5, 15, 22, 23, 26, 41

with hours of

lengthy amplification steps, the AAP-based method replaces complicated manipulations with simple washing and filtration, requiring only about 5 minutes. Furthermore, the sensitivity and specificity are ensured via the specific affinity of the aptamer against streptavidin. The excellent performance of this method in real biological samples is guaranteed by the combination of the allosteric effect of AAP and the enrichment of the fluorescence signal on SA beads. Therefore, we have obtained a good detection limit of 0.01 U/mL in buffer and 0.05 U/mL in cell lysates, which is comparable or superior to those of the latest unamplified detection methods.15, 24, 41-44 Finally, this method can be used to estimate the inhibition conditions of various inhibitors, making AAP an ideal platform for phosphorylation studies and inhibitor screening. To some extent, this method also manifests good versatility as long as performing alteration at the targetrecognition region. Compared with other DNA-based detection methods, AAP also needs to decrease the influence brought by several DNA-sensitive molecules for instance DNase. In brief, the AAP-based detection method without any signal amplification provides a convenient,


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sensitive and interference-free strategy for quantitative detection of T4 PNK, and shows great potential for phosphorylation studies, clinical therapy and drug screening.

ASSOCIATED CONTENT Supporting Information. Comparison of different methods for T4 PNK detection. (Table S1) This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *C Yang. E-mail: [email protected] * Z Zhu. E-mail: [email protected] * Y Song. E-mail: [email protected] ACKNOWLEDGMENT We thank the National Science Foundation for Distinguished Young Scholars of China (21325522), the National Science Foundation for Excellent Young Scholars of China (21422506), the National Natural Science Foundation of China (91313302, 21205100, 21275122, 21435004, 21521004), and the National Science Fund for Fostering Talents in Basic Science (NFFTBS, J1310024) for financial support. REFERENCES


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Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe

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