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Engineering a DNA-cleaving DNAzyme and PCR into a simplesensor for zinc ion detection

Jiacui Xu & Yanhong Sun & Yongjie Sheng & Yanqun Fei &Jin Zhang & Dazhi Jiang

Received: 8 January 2014 /Revised: 21 February 2014 /Accepted: 27 February 2014 /Published online: 28 March 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The development of a simple sensor (9NL27-Zn)based on DNAzyme and PCR and aimed at the detection oflow concentrations of zinc (II) ions is described. A specificZn(II)-dependent DNAzyme (9NL27) with DNA-cleavingactivity was employed. In the presence of zinc (II), theDNAzyme hydrolyzed DNA substrate into two pieces(5′ and 3′ fragments), forming 3′-terminal hydroxyl in the 5′fragment and 5′-phosphate in the 3′ fragments. Subsequently,the 5′ fragment left the DNAzyme and bound a short DNAtemplate. The 5′ fragment was used as a primer and extended asingle-stranded full-length template by Taq polymerase. Final-ly, this full-length template was amplified by PCR. Theamplified products had a quantitative relationship with Zn(II)concentration. Under our experimental conditions, the DNAsensor showed sensitivity (10 nM) and high specificity forzinc ion detection. After improvement of the DNA sensor, thedetection limit can reach 1 nM. The simple DNA sensor maybecome a DNA model for the detection of trace amounts ofother targets.

Keywords Sensor .DNAzyme . PCR .Zinc ion .C3-spacer .

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Introduction

DNA plays an essential role in biological systems because itcan store and transmit genetic information. However, in lab-oratories, DNA has the capacity for catalytic function, inwhich phosphodiester-cleaving activity has been most widelyinvestigated [1–5]. These catalytically active DNA are com-monly referred to as DNAzymes or deoxyribozymes. MostDNAzymes require metal ions for their activities, and someeven show metal specificity. Therefore, DNAzymes can beused for metal sensing. Many efforts have used DNAzymes askey components within analytical applications [6–10]. Liuet al. reported a fluorescent sensor for uranium [5]. Miaoet al. designed a colorimetric sensor for copper [11]. Zhanget al. engineered an electronic DNAzyme sensor for lead [12].In general, these DNA sensors contain at least two compo-nents: target recognition and signal amplification. A widelyused strategy is to link the target recognition portion(DNAzyme) closely with a signal amplification moiety suchas fluorophore, colorimetric or electrochemical signals.

The divalent zinc cation, Zn(II), is the second most abun-dant metal ion in the human body. Zn(II) has critical roles inthe cell both as a cofactor for many proteins (enzymes) and astructural element. This ion has important functions in thenervous, reproductive and immune systems and a central rolein development and growth. In recent years, a progress is therecognition that Zn(II) has functions as messengers in cellularinformation transfer, which affects proliferation, differentia-tion and apoptosis [13–16]. Thus, the highly sensitive andselective detection of Zn(II) ions is very important. Recently,several sensors of Zn(II) have also been proposed based ondifferent fluorophores (Table S1, Electronic SupplementaryMaterial) [17–21]. Obviously, these fluorescent sensors aremade up of organic molecules, which typically consist of aZn(II)-chelating unit, such as N,N-bis(2-pyridylmethyl)amine(BPA) or di(2-picolyl)amine (DPA), and a fluorescent unit.

Electronic supplementary material The online version of this article(doi:10.1007/s00216-014-7732-9) contains supplementary material,which is available to authorized users.

J. Xu :Y. Sun :Y. Sheng :Y. Fei : J. Zhang (*) :D. Jiang (*)Key Laboratory for Molecular Enzymology and Engineering of theMinistry of Education, College of Life Science, Jilin University,Qianjin Street 2699, 130012 Changchun, Chinae-mail: zhangjin@jlu.edu.cne-mail: jiangdz@jlu.edu.cn

Present Address:J. XuDepartment of Biochemistry, University of Wisconsin, Madison,WI 53706, USA

Anal Bioanal Chem (2014) 406:3025–3029DOI 10.1007/s00216-014-7732-9

Here, we report another strategy, DNAzyme-coupled PCR,to construct a DNA sensor (9NL27-Zn) for sensitive quanti-fication of the zinc ion. In the DNA sensor, a Zn2+-dependentDNAzyme was prepared for molecular recognition and PCRwas used for signal amplification.

Materials and methods

Materials

All standard and modified DNA oligonucleotides were pur-chased from Sangon Biotech (shanghai) Co., Ltd. (Shanghai,China) and purified by 8 % denaturing PAGE (Table S2,Electronic Supplementary Material). Metal ions, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid sodium salt(HEPES) and sodium chloride were purchased from SangonBiotech (shanghai) Co., Ltd. (Shanghai, China). Agarose waspurchased from Biowest. GelRedTM was purchased fromBiotium Inc. Ultrapure water from Milli-Q (Merck Millipore)was used in all of the experiments.

Detection of Zn2+ by using the 9NL27 DNAzyme

To detect zinc ions, the 50 nM DNAzyme and its 50 nMsubstrate were mixed in the buffer that contained a finalconcentration of 70 mM HEPES (pH 7.5) and 150 mM NaCl(10× stock solution was used). The solution was heated to 92 °Cfor 5 min and allowed to cool gradually to 37 °C in aMiniCycler (MJ Research Inc.). The reactions were initiatedby adding different concentrations of zinc ion and performedat 37 °C for 4 h.

PCR

PCR was performed on a MyCyclerTM thermal cycler (Bio-Rad Laboratories Inc.). The total volume of PCR mixture ineach EP tube was 50 μl containing 2 μl DNAzyme reactionmixture, 50 nM short template, 1 μM each primer, 0.1 unit/μlTaq DNA polymerase, 0.2 mM dNTPs, 1.5 mM MgCl2, and1× PCR buffer (10 mMTris-HCl (pH 8.8), 50mMKCl, 0.8‰NP-40). PCR products were achieved by thermal cycling forthe initialization step at 94 °C for 1 min; extension step at 94 °Cfor 20 s, 58 °C for 2 min and 72 °C for 2 min; amplificationstep at 31 or 36 (for the improvedDNA sensor) cycles at 94 °Cfor 20 s, 58 °C for 20 s and 72 °C for 20 s; and final elongationat 72 °C for 2 min. Finally, the PCR products were separatedby 2 % agarose gel electrophoresis. The amount of productthat accumulated was determined by staining with a fluores-cent dye (GelRedTM).

Results and discussion

Design of a DNA sensor (9NL27-Zn) for zinc ion detection

The 9NL27 DNAzyme is isolated via in vitro selection, whichis highly specific for Zn2+ and catalyzes DNA cleavage withformation of 5′-phosphate and 3′-hydroxyl (OH) DNA termini[22, 23]. The 3′-OH group can be recognized by Taq poly-merase for PCR amplification. Therefore, the 9NL27DNAzyme can be developed for molecular recognition toidentify Zn2+. The principles of the DNA sensor for analysingzinc ion are as follows (Fig. 1): In the presence of Zn2+, the9NL27 DNAzyme cleaves its DNA substrate into two pieces(5′ and 3′ fragments), forming a 3′-terminal OH group in the

Fig. 1 The general principle of a9NL27-Zn sensor by thecombination of a Zn2+-dependentDNAzyme and PCR. The redarrowhead indicates the cleavagesite of DNA substrate

3026 J. Xu et al.

5′ fragment. Subsequently, the 5′ fragment leaves the DNAzymeand binds a short DNA template to extend a single-strandedfull-length template by Taq polymerase. Finally, this full-length template was amplified by PCR. The amplified prod-ucts have a quantitative relationship with Zn2+ concentration.

To decrease the background signal, two strategies wereused: C3-spacer chemical modification and unpaired domainat the 3′-end of DNA, which can prevent Taq polymeraseextension. The designed DNAzyme, substrate and short tem-plate are 9NL27-C3, SLR-C3 and ST-12C3 (Table S2, Elec-tronic Supplementary Material). The substrate SLR-C3 con-sists of 3′-end SR and 5′-end SL. Only the SL fragment of thecleavage product can form full complementary base pairs withthe short template (ST-12C3), which would extend the full-length template (FT) by Taq polymerase. In addition, both ST-12C3 and 9NL27-C3 contain 12-nt unpaired domains (DL,WZ), which prevent error extension.

Sensitivity and specificity of the 9NL27-Zn sensor for Zn2+

The DNAzyme 9NL27-C3 and the substrate SLR-C3 wereincubated for 4 h under Zn2+ from 1 nM to 1 mM. Afterincubation, 2 μl of reaction product was used for PCR ampli-fication of 31 cycles. The FTwas generated through the shorttemplate (ST-12C3)-directed extension followed by the firstcycle of PCR, and the remaining 30 cycles were used toamplify the FT template, meaning signal amplification. Then,the PCR products were detected by gel electrophoresis.Figure 2 shows the rate of PCR amplification enhancementplotted against different Zn2+ concentrations from 1 nM to1 mM. The PCR products were highly dependent on theconcentration of Zn2+ used in the reaction mixture. At lowconcentrations of Zn2+ ranging between 1 and 50 nM, thePCR products increased slowly. As the concentration of Zn2+

increased from 50 nM to 50 µM, the products increasedsharply, then gently upon 50 µM. The detection limit of thesensor can reach 10 nM.

In the following, the selectivity of the 9NL27-Zn sensorwas determinedwith 11 different divalent metal ions includingMg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Ba2+, Pd2+

and Hg2+ at 1 mM concentrations. As shown in Fig. 3,9NL27-Zn only responded to zinc ions, which indicated goodselectivity. The sensor has appeared to be as selective as theDNAzyme upon which it is based.

Moreover, the experimental results (Fig. S1, ElectronicSupplementary Material) revealed that the 12-nucleotidelength of the unpaired DL sequence in the short templatewas important to decrease the background signal. When thelength of the DLwas shortened to 6, 3 and 0 nt (namely as ST-6C3, ST-3C3 and ST-C3, respectively, in Table S2, ElectronicSupplementary Material), the background progressively in-creased under the same condition, indicating that C3-spacermodification only at the 3′ end cannot efficiently prevent PCR

amplification because of incomplete chemical modification.Non-pair sequence extension at the 3′-end of the short

Fig. 2 Sensitivity of the 9NL27-Zn sensor for Zn2+ detection. The uppergraph is a gel electrophoresis image of the sensor toward Zn2+. PCRamplifications (P) increase over the background at varying Zn2+ concen-trations. The lower graph shows the rate of PCR amplification enhance-ment plotted against different Zn2+ concentrations from 1 nM to 1 mM.Inset: rates at the low Zn2+ region

Fig. 3 Selectivity of the 9NL27-Zn sensor. The upper graph is a gelelectrophoresis image of the DNA sensor for 11 different metal ions. Theconcentration of each metal ion is 1 mM. The lower graph shows PCRamplification (P) enhancement plotted against 11 different metal ions.The error bars are relative standard deviations from three repeatedexperiments

Engineering a DNA-cleaving DNAzyme and PCR into a simple sensor 3027

template was a simple and efficient way to reduce the back-ground signal.

The relationship between the incubation time andsignal amplification was investigated. Under single-turnover conditions, the kobs of the 9NL27 DNAzymeis 1.45 h−1, which is lower than that of the classicDNAzymes, such as 10-23 and 8-17 [2]. Therefore,we had to increase the incubation time. The sensor inFig. S2 (Electronic Supplementary Material) showedthat more sensitivity needed longer incubation time.However, an overlong incubation time would decreasethe practicability of the sensor.

Improved design of the 9NL27-Zn sensor

The C3-spacer can prevent Taq polymerase extension,and other modifications such as Spacer-18 also do that[5]. Spacer-18 was inserted in the DNA substrate (SLR-S18-C3) and short template (ST-S18-C3), and C3-spacermodified at the 3′-end of SLR-S18-C3 and ST-S18-C3.The 12 non-paired nucleotides (NPN) existed betweenspacer-18 and C3-spacer, which further decreased thebackground signal (Fig. 4A).

The results indicated that the new version of the sensorimproved the sensitivity of the Zn2+ ion down to 1 nM(Fig. 4B). Meanwhile, this sensor still kept its specificity,and no detectable product was formed in the presence of otherions (Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Ba2+,Hg2+and Pd2+) (Fig. 4C).

Recently, a new Zn(II) sensor, DA-ZP1-TPP, was reported[17]. Comparing with DA-ZP1-TPP and 9NL27-Zn, boththeir sensitivities can reach the level of nanomoles per litre.DA-ZP1-TPP shows a relatively lower fluorescence enhance-ment in the presence of Cd(II), while 9NL27-Zn only

responds to Zn2+. However, DA-ZP1-TPP can be targeted tomitochondria in live cells and detect free intracellular zincions. 9NL27-Zn works only with zinc ions in vitro.

Our sensor can be further improved and promoted based onthe following two aspects: molecular recognition and signalamplification. The 9NL27 DNAzyme carries a catalyticcore composed of 40 conserved nucleotides [22, 23],while the Breaker group recently has developed a highlyactive DNAzyme (I-R3) using Zn2+ as cofactor, havingonly 15 nucleotides in the catalytic core [24]. Using I-R3 DNAzyme instead of 9NL27 DNAzyme for mole-cular recognition will provide a smaller and more effi-cient sensor. In addition, PCR, a simple and cost-efficient approach for signal amplification, can beupgraded into quantitative PCR (qPCR) and digitalPCR (dPCR) to show a real-time signal, but higher-level instruments will be required [25].

Summary

The strategy of DNAzyme-coupled PCR has been dem-onstrated to have good compatibility [26, 27]. Herein,we use this strategy to engineer a DNA sensor for zincion detection. The specific Zn2+-dependent DNAzyme(9NL27) is employed to develop target recognition,while PCR were used as labels that amplify the sensingevent. This DNA sensor has been demonstrated for thesensitive and specific detection of zinc ions. Its majoradvantage is simple. All the equipments and experimen-tal conditions can be performed in most of bio-laboratories. This approach of the DNAzyme-coupledPCR is also adapted to other DNA-cleaving DNAzymeswhich can be engineered as a DNA sensor platform.

Fig. 4 Improved design of the 9NL27-Zn sensor by dual modifications. a Further improvement of the DNA sensor. Substrate: STR-S18-C3; shorttemplate: ST-S18-C3. b Sensitivity of the improved DNA sensor for Zn2+ detection. c Selectivity of the improved DNA sensor for Zn2+ detection

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Acknowledgments This research was supported by the FundamentalResearch Funds for Jilin University (200903096) and National NaturalScience Foundation of China (31100573).

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