Analytica Chimica Acta 844 (2014) 70–74

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A simple and highly sensitive DNAzyme-based assay for nicotinamide adenine dinucleotide by ligase-mediated inhibition of strand displacement amplification Cheng Jiang, Ying-Ya Kan, Jian-Hui Jiang *, Ru-Qin Yu * State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China

H I G H L I G H T S

G R A P H I C A L A B S T R A C T


The strategy carried out a visual assay for NAD+.
A strategy based on ligase-mediated inhibition on strand displacement amplification.
The colorimetric assay is simple, high sensitivity and high selectivity.
A novel platform for investigating cofactors, small molecules and DNA ligases.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 February 2014 Received in revised form 14 May 2014 Accepted 25 June 2014 Available online 10 July 2014

Existing strategies for detecting nicotinamide adenine dinucleotide (NAD+) or other cofactors are commonly cumbersome and moderate sensitive. We report a novel DNAzyme-based visual assay strategy for NAD+ based on ligase-mediated inhibition of the strand displacement amplification (SDA). In the presence of NAD+, the SDA can be inhibited by the ligase reaction of two primers, which can initiate the SDA reaction in the case of no ligation, resulting in a dramatically decreasing yield of the SDA product, a G-quadruplex DNAzyme that can quantitatively catalyze the formation of a colored product. Therefore, the quantitative analysis for NAD+ can be achieved visually with high sensitivity. The developed strategy provides a simple colorimetric approach with high selectivity against most interferences and a detection limit as low as 50 pM. It also provides a universal platform for investigating cofactors or other related small molecules as well as quantifying the activity of DNA ligases. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Nicotinamide adenine dinucleotide Inhibition Ligase-mediated Strand displacement amplification

1. Introduction Nicotinamide adenine dinucleotide (NAD+) is a cofactor widely existing in eukaryotic and prokaryotic organisms. Serving as a mediator of electron, NAD+ participates in many cellular metabolic processes. Additionally, it is also involved in some important

* Corresponding authors. Tel.: +86 731 88821916; fax: +86 731 88821916. E-mail addresses: [email protected] (J.-H. Jiang), [email protected] (R.-Q. Yu). http://dx.doi.org/10.1016/j.aca.2014.06.054 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.

biological functions including DNA repair [1], transcriptional regulation [2,3], calcium homeostasis [4], and cell apoptosis [5]. Studies have shown that the NAD+ level is interrelated with cancer, diabetes and neurodegenerative diseases [6–8]. Furthermore, NAD+ is found to be an essential substrate to some ligases, oxidoreductases, deacetylases, poly(ADP-ribose)polymerase and Sir2p family [9]. In view of its physiological significance, researchers have developed various methods for investigating NAD+ over the past decade. The reported methods were mainly based on enzyme cycling [10], high-performance liquid chromatography (HPLC) [11],

C. Jiang et al. / Analytica Chimica Acta 844 (2014) 70–74

capillary electrophoresis [12], nuclear magnetic resonance (NMR) [13], electrospray ionization mass spectrometry (ESI-MS) [14], electrochemistry [15] and molecular fluorescence [16,17]. However, enzyme cycling is a rather complicated assay system and requires highly purified enzyme which is susceptible to the detection conditions like pH and redox potential. Additionally, HPLC, NMR and ESI-MS require expensive instruments, and electrophoresis analysis could not provide sufficient sensitivity. Low detection limit could be obtained by the cost-effective electrochemical strategy, but the electrochemical detection is a heterogeneous method in which the variance between parallel electrodes is commonly difficult to be controlled. Although the fluorescent-labeled methods could circumvent some limitations of other strategies and exhibited nice accuracy and sensitivity for analyzing NAD+, they always require complicated chemical modifications using various fluorescent dyes. Therefore, the development of simple and highly sensitive homogeneous sensors for NAD+ is still desirable. There are two possible ways to improve the performance of sensors: constructing an efficient signal transducer and using a further signal amplification strategy. G-quadruplex DNAzyme is an efficient signal medium which has been widely applied in various label-free analytical systems [18–22]. These sensors could provide acceptable sensitivity for detecting heavy metal ions or biomolecules by using the catalytic ability of the G-quadruplex–hemin complex with horseradish peroxidase (HRP) mimicking activity. The DNAzyme-based methods can further improve their detection sensitivity by combining nucleic acid amplification strategies such as strand displacement amplification (SDA) and rolling circle amplification [23–26]. For example, the performance of DNA mutation assay has been improved by orders of magnitude by utilizing SDA [18,23]. Although SDA technique could produce numerous DNAzyme fragments by the circularly polymerizing and nicking [27], integrating SDA to the sensor for small molecules remains largely unexplored. Herein, we propose to integrate SDA technique into the simple colorimetric assay to realize a novel sensing platform for small molecules such as NAD+ with enhanced sensitivity. The sensing platform is constructed mainly using three linear DNAs, in which one is the template DNA used to produce HRP-mimicking DNAzyme and the other two are the primers used in the templated ligation reaction. In the presence of NAD+, the two primer DNAs are ligated to an integrated primer with the assistance of the DNA template. The integrated primer formed in the presence of NAD+ would inhibit SDA because its 30 mismatched bases cannot guide the polymerase extension. In the absence of NAD+, the extension process can proceed to produce a great amount of G-quadruplex DNAs via SDA. Thus, the concentration of NAD+ is correlated to the yield of the G-quadruplex DNA, which complexing with hemin and forming a HRP-mimicking DNAzyme, can catalyze the oxidation of 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonate dianion) (ABTS2) to a colored product. Thus, the ligase-mediated strategy can realize the quantitative assay of NAD+ by visualizing the color change and by absorption spectral detection. 2. Experimental 2.1. Reagents and instruments Escherichia coli (E. coli) DNA ligase, Nt.BbvC I, and Klenow fragment (30 -50 exo) were purchased from New England Biolabs, Inc., (Beverly, MA, USA). Nicotinamide adenine dinucleotide (NAD + ), the reduced form of nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide phosphate hydride (NADPH), adenosine triphosphate (ATP), adenosine, adenosine diphosphate

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(ADP), adenosine monophosphate (AMP) and the synthetic oligonucleotides were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). ABTS, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonate (HEPES), tris(hydroxymethyl)aminomethane (Tris) and hemin were purchased from Sigma–Aldrich (St. Louis, USA). All other chemicals were of analytical reagent grade and used without further purification. Ultrapure water from a Millipore filtration system (resistance >18.2 MV) was sterilized before use. The designed DNA sequences were as follows: DNA1, 50 -TCT GTA GTG A-30 ; DNA2, 50 -p-GAA GTC TAT GTT-30 ; DNA3, 50 -CCC AAC CCG CCC TAC CCG CTG AGG CAT AGA CTT CTC ACT ACA GA-30 ; DNA control (DNAc), 50 -TCT GTA GTG AGA AGT CTA TGT T-30 . Ultraviolet absorption spectra were recorded from 400 to 500 nm using a Shimadzu UV-2450 spectrophotometer with HYPER UV version 1.50 software. The pH measurements were performed with a Mettler-Toledo FE20 pH meter. 2.2. Ligation reaction and strand displacement amplification The 10 buffer (500 mM NaCl, 100 mM Tris–HCl and 100 mM MgCl2, pH 7.9) firstly was prepared and diluted before use in ligation reaction and SDA process. An annealing mixture containing 1 mM of DNA1, 1 mM of DNA2 and 1 mM of DNA3 was prepared using 10 buffer. To make DNA1 and DNA2 hybridize with DNA3, the mixture was heated to 90  C for 5 min and cooled to room temperature. The ligation reaction was performed at 37  C for 30 min in a volume of 20 mL, which is composed of 10 mL annealing mixture, 0.2 U mL1 of E. coli DNA ligase and varying concentrations of NAD+. After ligation reaction, 1 mL of dNTPs (10 mM), 0.5 mL of Klenow polymerase (5 U mL1), and 1 mL of Nt.BbvC I nickase (10 U mL1) and 2.5 mL of 2 buffer were added; finally, SDA was performed at 37  C for 60 min in a reaction solution of 25 mL, which consists of 10 mM Tris–HCl (pH 7.9), 50 mM NaCl and 10 mM MgCl2. 2.3. Colorimetric assay To make all reaction processes perform in a centrifuge tube, 10 mL of 10 HEPES buffer (250 mM HEPES, 2 M NaCl, 100 mM KCl, and 0.5% Triton X-100, pH 5.2), 1.25 mL of hemin (20 mM) and 63.75 mL of H2O were then added to the mixture of SDA reaction. The resulting mixture was incubated at 37  C for 30 min where G-quadruplex DNA and hemin could form G-quadruplex–hemin complex with the HRP-mimicking activity. Finally, the G-quadruplex DNAzyme activity was measured according to the color change from colorless ABTS2 to colored ABTS
. The corresponding absorption spectra were recorded at 420 nm in 10 min after the addition of 10 mL of 10 mM ABTS2 and 10 mL of 40 mM H2O2. 2.4. Selectivity evaluation The analogs or interfering substances of NAD+, including NADH, NADP, NADPH, ATP, ADP and AMP were also analyzed using the identical procedure to evaluate their interfering effect on the assay of NAD+. The final concentration of the analogs in the ligation reaction solution was fixed to 50 nM. Their absorption intensities were measured respectively, and the interfering effect was evaluated by comparing the absorption change caused by NAD+ with that caused by interfering substances. 3. Results and discussion 3.1. Principle of DNAzyme-based assay for NAD+ based on the ligasemediated inhibition on SDA The developed strategy for detecting NAD+ is illustrated in Scheme 1. A DNA duplex structure is firstly obtained by hybridizing

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Scheme 1. Schematic illustration of the DNAzyme-based assay NAD+ for by the ligase-mediated inhibition on SDA.

DNA1 and DNA2 with DNA3. Here DNA2 contains a phosphorylated 50 end necessary for ligase reaction and a mismatched twothymine end at 30 terminus designed to inhibit polymerase extension of the ligated primer [28,29]. DNA3 consists of four regions, I, II, III and IV, corresponding to the complementary sequence of DNA1 and DNA2, the recognition region of nickase Nt. BbvC I (who can recognize and nick the 50 -CC!TCAGC-30 sequence contained in the polymerized dsDNA. The symbol ‘!’ represents the nicking site) and the template of G-quadruplex DNA. In the absence of NAD+, DNA1 can act as the primer to initiate SDA, resulting in a great amount of G-quadruplex DNA. In the presence of NAD+, DNA1 is ligated to DNA2 and the efficient extension by the primer DNA1 is inhibited, which induces a decreased yield of G-quadruplex DNA. Complexing with hemin and forming a HRPmimicking DNAzyme, the G-quadruplex DNA can catalyze the oxidation of 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonate dianion) (ABTS2) to a colored product, which allows a visualized detection of the amount of G-quadruplex DNA and indirectly indicates the concentration of NAD+. Based on the amplification effect of the ligase-mediated SDA, the developed strategy provides a simple but very sensitive and selective colorimetric approach for NAD+ detection. To demonstrate the feasibility of this strategy, the absorption spectra of the resulting solutions in the presence and/or absence of E. coli DNA ligase and NAD+ were collected and their peak intensities at 420 nm were recorded respectively and compared with each other. As described in Fig. 1, both curve a and curve b show a strong absorption in the presence of only NAD+ or E. coli DNA ligase. It demonstrates that the separate addition of NAD+ or E. coli DNA ligase does not show inhibiting effect on SDA companying with production of plenty of G-quadruplex DNAs. The slightly lower absorption intensity of the sample ‘b’ as compared to that of the sample ‘a’ could be attributed to the fact that the stock buffer of E. coli DNA ligase used to contain few reducing substances which might affect the chromogenic reaction catalyzed by G-quadrplex DNAzyme. By contrast, curve c exhibits a very low absorption intensity, which indicates that DNA1 and DNA2 are connected to an integrated primer in the presence of both NAD+ and E. coli DNA ligase and the SDA was obviously inhibited. To further confirm the lower absorption signal is caused by the inhibiting effect of the integrated primer, the synthetic oligonucleotide (DNAc) is used as the substitute primer for DNA1 and DNA2 in SDA without the addition of NAD+ and E. coli DNA ligase. Compared with curve c, curve d presents a similar absorption intensity that verifies the ligase-mediated inhibiting effect on SDA. Besides, the inset of Fig. 1 is the photograph of the corresponding samples, which indicates that the present strategy could offer a visible assay for NAD+ by naked eye.

Fig. 1. The absorption spectra of different samples in the presence of (a) 30 nM NAD + , (b) 0.2 U mL1 E. coli ligase, (c) 30 nM NAD+ + 0.2 U mL1 E. coli ligase, (d) 50 nM DNAc. These samples are all treated by the same SDA procedure, and the combination of DNA1 and DNA2 is replaced by DNAc in the sample ‘d’.

3.2. The optimization of experiment conditions The concentration of DNAs used in this sensing system is an important factor to achieve an optimal performance. Referring to previous report [30], high concentration of primer and template could have a negative effect on the polymerization efficiency of SDA and too low concentration could cause problems in detecting the high concentration of analytical target. The signal to noise (S/N) ratio from four groups of DNA (DNA1, DNA2 and DNA3 in equal concentration) were investigated in the absence and presence of NAD+. The results in Fig. 2A show that the signal and background absorption intensities both increased with the increasing concentration of DNA group and the maximum S/N ratio was observed for the concentration group of 50 nM, therefore, the latter is chosen as the most suitable concentration of the assay. The effect of ligation reaction time was studied. According to the data shown in Fig. 2B, the change of the absorption intensity of the resulting solution increased with increasing time length within 30 min in the presence of the same concentration of NAD+ and reached a maximum value at 30 min. Thus, 30 min was used as the optimal time length of ligation reaction in the following experiments. 3.3. Quantitative detection of NAD+ Under the optimized conditions, a series of NAD+ concentrations were analyzed to evaluate the performance of the proposed strategy. The ultraviolet absorption spectra for different concentrations of NAD+ are illustrated in Fig. 3A, the absorption intensity decreases with the increasing concentration of the target in the range from 0 to 50 nM. Furthermore, the absorption intensity reduced to a minimum value when the concentration of NAD+ reached 50 nM, meaning that SDA is almost inhibited completely and few DNAzyme was produced when enough NAD+ was added. As shown in Fig. 3B, the absorption intensity was found to be linear related with the concentration of NAD+ in the range from 0.05 nM to 2 nM. The regression equation is depicted as ‘y = 1.27–0.20x’ and the correlation coefficient is 0.992. The detection limit (DL) of the approach was calculated as 50 pM according to the 3srules. The corresponding limit of quantification (LOQ) is 0.42 nM (the DL and the LOQ corresponding to the x value when y = Ablank (the absorbance of the blank sample)-3s (the standard deviation of Ablank) and y = Ablank-10s, respectively). The detection limit is about

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Fig. 2. (A) The effect of the concentration of different DNA groups on the signal to background ratio, (B) the effect of the reaction time of ligase on the change of the absorption intensity of the NAD+ sensing system. The concentration of NAD+ is 30 nM. Error bars were the standard deviation of three measurements.

100 times lower than those of early enzyme cycling assay [10] and the recent fluorescence analysis based on G-quadruplex assembling [31]. Compared with the two methods, the developed strategy presents one or a few advantages as follows: colorimetric measurement, simple procedure and no requirement of highly purified enzyme. Above comparison proves that the SDA-based strategy of signal amplification could be successfully employed in quantifying NAD+ with improved sensitivity. Our detection limit is equal or even higher compared to that of the ligation-triggered DNAzyme cascade fluorescence assay [17] or the enzymatic assay based on poly(ADP-ribose)polymerase-1 (PARP-1) [32]; however, the present strategy is a convenient non-fluorescent method as well as a universal platform applying to detect other small molecular cofactors. In order to demonstrate the possible application of the developed strategy in biological matrix, the absorption intensities toward various concentrations of NAD+ prepared in 1% human serum were investigated. The results indicate (Fig. S1 and Table S1) that the assay based on ligasemediated inhibition of strand displacement amplification is promising to be applied in real samples containing complex biological matrices. 3.4. The selectivity of the assay The selectivity of the assay with respect to small molecules is vital to evaluate the practicability of a sensing system. Some early

methods for detecting NAD+ were based on the conversion of NAD+ into its derivatives (such as NADH), thus, they suffered the inherent drawback caused by the difficulties in discriminating NAD+ from NADH or other analogs. Fortunately, a strategy based on the enzymatic reaction solved the problem by using the high dependency of enzyme activity and its cofactor. The experimental result of the selectivity investigation is shown in Fig. 4, in which the changes of the absorption intensities corresponding to several interfering substances were evaluated under identical analytical conditions. Only a negligible change of intensity was observed compared to the response of NAD+ when 50 nM of NADH, NADP, NADPH, ATP, ADP or AMP were added to the developed sensing system. It clearly indicates that the developed ligation-mediated sensing system possesses high selectivity for detecting NAD+ and has an excellent ability to distinguish NAD+ from its analogs. 4. Conclusions In summary, a simple, highly sensitive strategy for detecting NAD+ has been developed by combining the inhibition of the 30 -unmatched primer on SDA with the efficient catalytic ability of G-quadruplex DNAzyme. The sensing system has three significant advantages: firstly, it possesses high selectivity toward NAD+ and could distinguish the analytical target from other small molecules or its analogs; secondly, the DNAzyme-based strategy carries out a visible assay for NAD+ based on the chromogenic reaction

Fig. 3. (A) The absorption spectra recorded for different concentrations of NAD+ with the DNAzyme-based sensor and (B) the absorption intensity versus the NAD+ concentration plot in the range from 0 to 50 nM. Inset is the calibration curve for NAD+ with the concentration ranging from 0.05 to 2 nM. Error bars were the standard deviation of three measurements.

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Fig. 4. Selectivity of the DNAzyme-based sensing system for NAD+. The detection concentration of NAD+ is 30 nM and those of all interfering substances are 50 nM. The change of the absorption intensities is evaluated under identical experimental conditions. Error bars are the standard deviation of three measurements.

catalyzing by G-quadruplex DNAzyme, thus making the quantitative information of NAD+ conveniently observed by the naked eye; thirdly, SDA is further employed for the signal amplification by the combined action of nickase and polymerase, resulting in a high sensitivity with a detection limit of 50 pM for NAD+ far below those of previously reported methods. Furthermore, the proposed strategy based on the inhibition on SDA is a universal analytical system, thus provides a novel avenue for investigating cofactors or other related small molecules as well as quantifying the activity of DNA ligases. Acknowledgements This work was supported by NSFC (21275002, 20905022, 21025521, 21035001, 21190041), National Key Basic Research Program (2011CB911000), NSF of Hunan Province (10JJ7002) and Scientific Research Fund of Hunan Provincial Education department (08A065). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.06.054. References [1] A. Wilkinson, J. Day, R. Bowater, Bacterial DNA ligases, Mol. Microbiol. 40 (2001) 1241–1248. [2] S.-i. Imai, C.M. Armstrong, M. Kaeberlein, L. Guarente, Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase, Nature 403 (2000) 795–800. [3] Q. Zhang, D.W. Piston, R.H. Goodman, Regulation of corepressor function by nuclear NADH, Science 295 (2002) 1895–1897. [4] A.H. Guse, X. Gu, L. Zhang, K. Weber, E. Krämer, Z. Yang, H. Jin, Q. Li, L. Carrier, L. Zhang, A minimal structural analogue of cyclic ADP-ribose: synthesis and calcium release activity in mammalian cells, J. Biol. Chem. 280 (2005) 15952– 15959. [5] J. Luo, A.Y. Nikolaev, S.i. Imai, D. Chen, F. Su, A. Shiloh, L. Guarente, W. Gu, Negative control of p53 by Sir2a promotes cell survival under stress, Cell 107 (2001) 137–148. [6] F. Torabi, K. Ramanathan, P.-O. Larsson, L. Gorton, K. Svanberg, Y. Okamoto, B. Danielsson, M. Khayyami, Coulometric determination of NAD+ and NADH in normal and cancer cells using LDH, RVC and a polymer mediator, Talanta 50 (1999) 787–797.

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