G-quadruplex based two-stage isothermal exponential amplificationreaction for label-free DNA colorimetric detection

Ji Nie, De-Wen Zhang, Cai Tie, Ying-Lin Zhou n, Xin-Xiang Zhang n

Beijing National Laboratory for Molecular Sciences (BNLMS), MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry,Peking University, Beijing 100871, PR China

a r t i c l e i n f o

Article history:Received 12 December 2013Accepted 17 January 2014Available online 24 January 2014

Keywords:G-quadruplexDNA detectionIsothermal exponential amplificationLabel-freeColorimetry

a b s t r a c t

A novel G-quadruplex based two-stage isothermal exponential amplification reaction (GQ-EXPAR) wasdeveloped for label-free DNA colorimetric detection in this work. The exponential amplified trigger DNAin the first stage can convert into G-quadruplex sequence EAD2 by a linear amplification circuit in thesecond stage. Created EAD2 can form G-quadruplex/hemin DNAzyme to act as a direct signal readoutelement. The GQ-EXPAR combines the exponential amplification of DNA sequence and the peroxidase-mimicking DNAzyme induced signal amplification, which achieves tandem dual-amplification. Takingadvantages of isothermal incubation, this label-free homogeneous assay obviates the need of thermalcycling . As no complex synthesis or extra downstream operation is needed, the whole easy handlingprocedure can be finished in no more than 1 h. This assay allows the sensing of the model DNA with thelimit of detection to be 2.5 pM. Moreover, it demonstrates good discrimination of mismatched sequences.The strategy has also been successfully implemented to sensitively detect Tay–Sachs genetic disordermutant.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

DNA detection is critical for accurate examination of clinicalpathogen and the early diagnosis of cancer or genetic disease(Sassolas et al., 2007). An ideal assay should be rapid, real-time,simple and sensitive for quantification. It is ordinarily performedby amplifying trace amounts of target oligonucleotide to detect-able levels. The most famous nucleic acid amplification is thepolymerase chain reaction (PCR) (Saiki et al., 1988), which relies ontemperature cycling protocol and polymerase activity. However,precision thermal cycling among three temperatures imposesinstrument constraints. Many isothermal amplification techniqueshave emerged as alternatives with excellent performance, such asrolling circle amplification (RCA) (Lizardi et al., 1998), loop-mediated amplification (LAMP) (Hsieh et al., 2012; Notomi et al.,2000), strand displacement amplification (SDA) (Connolly andTrau, 2010; Walker et al., 1992), helicase-dependent amplificationreaction (HDA) (Huang et al., 2011; Vincent et al., 2004), hybridi-zation chain reaction (HCR) (Dirks and Pierce, 2004; Dong et al.,2012) and nucleic acid sequence-based amplification (NASBA)(Compton, 1991). While proceeding at constant temperature,

isothermal nucleic acid amplification shows more potential inrealizing low-cost point of care molecular diagnosis.

EXPAR (isothermal exponential amplification reaction) firstreported by Galas' group is an isothermal molecular chain reactioncombining polymerase strand extension and single strand nicking(Van Ness et al., 2003). It can synthesize short oligonucleotideswith high amplification efficiency and rapid amplification kineticsdue to the DNA biological circuit with feedback design. Accordingto the number of templates and circuits involved, EXPAR in nucleicacid detection contains two main modes, one-stage and two-stage: (1) in the one-stage mode, the most basic and originalEXPAR, real-time fluorescence PCR machine is used for monitoringthe generation of partially or completely dsDNA (double-strandedDNA) (Jia et al., 2010). As the fluorescent intensity is correlated tothe amount of double-stranded regions, the point of inflection isused for quantification. Also, a kind of single-stranded longtemplate is developed, which can create trigger and reporteroligonucleotides simultaneously. Extra time is needed to recordthe fluorescence signal via growing nanoclusters (Liu et al., 2012;Wang et al., 2013b) or achieve satisfactory detection limit (Wanget al., 2013a); (2) in the two-stage mode, DNA sequence amplifica-tion and conversion are two divided stages. The created single-stranded reporter oligonucleotides converted in the second stagecan bridge DNA functionalized gold nanoparticles into aggregation(Tan et al., 2005) or hybridize with labeled DNA capture andreporter probes for quantum dot based fluorescence resonanceenergy transfer (Zhang and Zhang, 2012). The signal is directly

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0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.bios.2014.01.032

n Corresponding authors. Tel./fax: þ86 1062754112.E-mail addresses: zhouyl@pku.edu.cn (Y.-L. Zhou),

zxx@pku.edu.cn (X.-X. Zhang).

Biosensors and Bioelectronics 56 (2014) 237–242

correlated to the single-stranded reporter DNA produced in EXPARcircuit. Sophisticated equipment, time-consuming synthesis ofnano-material or multistep operation after amplification reactionto achieve signal readout limits their further application in thesimple and rapid genetic diagnosis.

Treating color changes as signal readout might make elabor-ated DNA biosensors more intuitionistic and convenient withoutlosing reliability and accuracy. Herein, we first developed a novelG-quadruplex based two-stage isothermal exponential amplificationstrategy for label-free DNA colorimetric detection. G-quadruplex is ahigher-order nucleic acid nano-structure generated from repetitiveG-rich sequence motifs. It can form G-quadruplex/hemin complexin the presence of hemin and mimic horseradish peroxidase activity(Cheng et al., 2009). Acting as a tag with catalytic activity for signaltransduction, G-quadruplex can be flexibly designed into nucleicacid biosensors for label-free homogeneous amplification (Zhaoet al., 2013). In our strategy, the exponential amplified targetoligonucleotides in the first stage can convert into G-quadruplexsequences EAD2 for DNAzyme-based colorimetry by a linear ampli-fication circuit in the second stage. Two kinds of amplificationprocedures combined into the system, the exponential amplificationof DNA sequence via EXPAR and the G-quadruplex/hemin mimic-peroxidase induced signal amplification, provide highly amplifiedefficiency and sensitive quantification. As no complex synthesis orextra downstream operation is needed, the developed assayachieves simple, rapid, sensitive, label-free and low-cost detectionof DNA.

2. Experimental section

2.1. Reagents and apparatus

All HPLC-purified DNA oligonucleotides (listed in Table S1) weresynthesized by Sangon Biological Engineering Technology & ServicesCo., Ltd. (Shanghai, China). The vent (exo-) DNA polymerase andnicking endonuclease Nt.BstNBI were purchased from New EnglandBiolabs (Beverly, MA). TMB �2HCl (TMB: 4,40-diamino-3,30,5,50-tetra-methylbiphenyl) was purchased from Ameresco (USA). Hemin wasobtained from Sigma-Aldrich (St. Louis, MO, USA). It was prepared indimethyl sulfoxide (DMSO) and stored at �20 1C as 5 mM stocksolution. All other reagents were at least of analytical reagent grade.Human serum samples were kindly provided by Peking UniversityHospital (Beijing, China). Absorption signals were recorded ona Thermo Scientific Multiskan FC Microplate Photometer (USA).

Temperature gradient optimization was carried out by a TC-512Gradient PCR (TECHNE, UK).

2.2. GQ-EXPAR assay

The reaction mixtures for GQ-EXPAR were prepared separatelyas part A and part B. Part A consisted of X0–X0 template, target DNAX, Nt.BstNBI buffer and dNTP. Part B consisted of X0–Y0 template,the nicking endonuclease Nt.BstNBI, vent (exo-) DNA polymeraseand ThermoPol buffer. Part A and Part B were mixed immediatelycontaining X0–X0 template (200 nM), X0–Y0 template (300 nM),dNTP (375 μM), Nt.BstNBI (0.8 U/μL), vent (exo-) DNA polymerase(0.1 U/μL), different concentrations of target DNA X, 1� ThermoPolbuffer (20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4,2 mMMgCl2, 0.1% Triton X-100) and 0.5�Nt.BstNBI buffer (25 mMTris–HCl, pH 7.9, 50 mM NaCl, 5 mM MgCl2, 0.5 mM DTT). Themixture at a volume of 10 μL was incubated at 55 1C for 30 min andquenched at 4 1C. In spiked bio-sample analysis, different concen-trations of target oligonucleotides were spiked with 20% humanserum and detected following the above procedure. In TS-mutantdetection, the reaction was performed at 58 1C for 1 h.

After the amplification reaction, 4 μL of 50 μM hemin and 14 μL1�ThermoPol buffer were added. A 5 μL resulting solutiondropped into 96-well plate was mixed with 100 μL TMB–H2O2

solution (0.12 mg/mL TMB �2HCl, 18 mM H2O2, 0.1 M NaH2PO4–

Na2HPO4, pH 6.0) to start the chromogenic reaction. G-quadru-plex/hemin DNAzyme can catalyze H2O2-mediated oxidation ofTMB. After 3 min incubation at room temperature (avoiding directlight exposure), the reaction was terminated by addition of 50 μL2 M H2SO4. Then photographs were taken or the absorbance wascollected as (A450–A620) a dual wavelength mode by a MicroplatePhotometer.

3. Results and discussion

3.1. The principle of GQ-EXPAR

This GQ-EXPAR integrates two stages (Scheme 1): the firststage is an exponential amplification with template X0–X0 fortarget X. The second stage is a linear amplification via templateX0–Y0 which enables the conversion of amplified trigger X to areporter Y for further colorimetry. The template X0–X0 consists oftwo repeated complementary sequences of X separated by thecomplementary of nicking enzyme recognition site (50-GAGTC-30)

Scheme 1. Schematic principle of GQ-EXPAR. (Inset) The detail of nicking enzyme recognition site.

J. Nie et al. / Biosensors and Bioelectronics 56 (2014) 237–242238

and cleavage site (four bases downstream). After trigger X primingX0–X0 template, the primer-template can be extended by vent(exo-) polymerase at 55 1C. Then, the key to achieve circuitcirculating, nicking enzyme Nt.BstNBI cleaves a phosphodiesterbond to create an oligonucleotide that either melts off or isdisplaced from template X0–X0. After dissociation, the regeneratedprimer-template can continue to produce oligonucleotide X in amanner of linear amplification. Created trigger X then primesother template X0–X0 leading to exponential amplification circuit.X can also prime a second amplification template X0–Y0 to triggerthe second stage, the conversion of amplified X to signal reporterY, EAD2 (a G-quadruplex sequence) in a linear amplification. EAD2can self-assemble G-quadruplex sequence into active DNAzymewith hemin. Subsequently, it catalyzes colorimetric reaction ofTMB–H2O2, which provides amplified optical signals. Exponentialand linear amplification combined two-stage EXPAR and G-quad-ruplex/hemin DNAzyme amplification are the core of GQ-EXPAR,which actually achieves tandem dual-amplification. Rapid anddirect monitoring of converted reporter EAD2 gets rid of tediousdownstream operations for signal readout. Also, it avoids signalinterference from non-specific background amplification inducedby spontaneous extended dsDNA, which may cause significantbackground interference in assays based on dsDNA intercalativefluorescent dye.

3.2. Feasibility of GQ-EXPAR for DNA detection

To evaluate the feasibility of GQ-EXPAR, a 22nt DNA sequencewas used as a model to perform several controls. No polymerasemeans no extension to the transient primer/template duplex.Nicking enzyme makes no sense to the partly complementaryduplex without dsDNA recognition and cleaving site. In contrast,with polymerase only, an ideally 1:1 stoichiometry of target X toelongated dsDNA is obtained. No nicking means no created newtrigger X or reporter Y and no continuous circulation. As shown inFig. 1(A), it was clear that the strategy was dependent on both thevent (exo-) polymerase and nicking enzyme Nt.BstNBI, and nosignal increase was observed when either of them was omitted.

We also tested the systemwith and without template X0–X0, whichwas the key to develop a feedback circuit for exponential increas-ing. There only exists an X0–Y0 linear amplification withouttemplate X0–X0. In the presence of 100 pM trigger X and X0–Y0,color change was not obvious (Fig. 1(B)). (We had tested theperformance of X0–Y0 linear amplification under the same condi-tion, and above 10 nM target X was detectable, data not shown.)However, with both X0–X0 and X0–Y0 templates forming a completeGQ-EXPAR circuit, 100 pM target X triggered a remarkable colori-metric signal. To investigate the specificity, one hundred times ofrandom sequences were added instead of trigger X and notriggering event had happened (Fig. S1). The correspondingphotographs analyzed by agarose gel electrophoresis are illu-strated in Fig. S2.

3.3. Optimization of the experimental conditions

As target X can competitively hybridize with both X0–X0 andX0–Y0, the ratio of templates X0–X0 and X0–Y0 is a fatal factor toinfluence the production of reporter Y. With higher molar ratios ofX0–X0, the trigger X was sufficiently amplified by an exponentialcircuit in the first stage but not converted to reporter Y effectively;with lower molar ratios of X0–X0 the exponential feedback circuitwas hindered, which subsequently affected the quantity of repor-ter Y created in the second stage. Keeping the total concentrationof templates X0–X0 and X0–Y0 to be 500 nM, the ratios of 1:4, 2:3,3:2, and 4:1 were quantitatively evaluated (Fig. 2A). The levels ofsignal increasing were calculated to be 11.8%, 185.8%, 156.4% and12.0%, successively. Therefore, the ratio of 2:3 was selected fortemplate X0–X0:X0–Y0 as the optimal condition.

Amplification time is another essential factor to determine theperformance of GQ-EXPAR. To avoid the signal increase induced bynonspecific background amplification in the absence of target X,such as the generation of reporter Y from spurious triggeringevent, an optimal 30 min was selected for GQ-EXPAR incubationunder the optimal ratio of templates (Fig. 2B). Within the samegiven time, more initial target X can trigger more amplification

Fig. 1. (A) The influences of Nt.BstNBI and Vent (exo-) DNA polymerase on GQ-EXPAR performance. All the solutions contained template X0–X0, X0–Y0 and 10 nM target X.(B) The control experiments of GQ-EXPAR performed without target X, without amplification template X0–X0 and with 100 pM target X, individually.

J. Nie et al. / Biosensors and Bioelectronics 56 (2014) 237–242 239

template and lead to more exponential circuit, which indicatesmore conversion to reporter EAD2.

3.4. The performance of GQ-EXPAR for DNA detection

The quantification performance of GQ-EXPAR for target Xdetection was investigated under the above optimal conditions.The absorption values A450–A620 after termination of the TMB–H2O2 coloration were recorded in the presence of different targetDNA concentrations. As shown in Fig. 3(B), the colorimetricresponses increased when the target was raised from 0 to10 nM. This indicated that the amount of created reporter EAD2was highly dependent on the concentration of trigger X. Even10 pM target X triggered remarkable color change that could beeasily distinguished by naked eyes (Fig. 3A). Target X could bequantified ranging from 10 pM to 10 nM with the limit of detec-tion (LOD) estimated to be 2.5 pM (25 amol in 10 μL) based on asignal-to-noise ratio (S/N) of 3.

The specificity of the proposed strategy was further investi-gated by perfect-matched target X, single-base mismatched, two-bases mismatched and three-bases mismatched DNAs (Fig. 4). Thesignal responses of two-bases and three-bases mismatched DNAs

show no difference to that of the negative control (in the absenceof target X). The single-base mismatched DNA shows 19.3% signalincrease, which is about one-fifth of that triggered by the perfect-matched target X (Fig. 4B). We found that mismatched DNAs were

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Fig. 2. (A) Variation of the level of signal increase as a function of the molar ratio ofX0–X0:X0–Y0 . The total concentration of X0–X0 and X0–Y0 was kept at 500 nM. Level ofsignal increase¼(Y�N)/N�100%, where Y is the signal triggered by 1 nM target Xand N is the signal of corresponding negative control in the absence of target X.(B) Variation of the level of background increase at different incubation times in theabsence of target X. Experimental conditions: template X0–X0 and X0–Y0 concentra-tions were fixed to 200 nM and 300 nM, respectively.

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Fig. 3. (A) The photograph for the colorimetric detection of target X by naked eyes.(Negative control: detection reaction without target X) (B) Calibration curve ofsignal response (A450–A620) vs. target X concentrations. Signal responses wereobtained via monitoring dual wavelengths after termination reaction. The experi-ment was performed under optimal conditions.

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Fig. 4. (A) The photograph of specificity evaluation for perfect-matched target X,single-base, two-bases and three-bases mismatched sequences. (B) The level ofsignal increase of the GQ-EXPAR assay for analyzing 100 pM perfect-matched targetX, single-base, two-bases and three-bases mismatched sequences under the sameexperimental condition, individually. Level of signal increase¼(Y�N)/N�100%,where Y is the signal triggered by 100 pM DNA sample and N is the signal ofcorresponding negative control.

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more difficult to initialize the GQ-EXPAR. Only perfect-matchedtarget X can trigger remarkable colorimetric response (Fig. 4A).Thus, our strategy exhibited good performance in the discrimina-tion of mismatched sequences.

To evaluate the feasibility of the GQ-EXPAR assay for morecomplex sample, we carried out the quantification performance byspiking target DNA into human serum. Also, 10 pM target X cantrigger notable color change. In Fig. S3, a calibration curve isachieved with LOD calculated to be 4.6 pM (S/N¼3). This indicatedthe potential of applying our strategy for DNA detection in acomplicated biological matrix.

3.5. The generalization of GQ-EXPAR

In order to test the general applicability of GQ-EXPAR wedesigned, the platform was further implemented to the detectionof Tay–Sachs genetic disorder mutant. Tay–Sachs disease is aninherited disorder that leaves the body unable to produce hex-osaminidase A for ganglioside GM2 degradation (Lu et al., 2012;Wang et al., 2012). Since the target sequence was increased to25 nt length, several factors of GQ-EXPAR were adjusted. Thermalproperties of DNA oligonucleotide, diffusion and annealing oftrigger X to template, activity of thermophilic DNA polymeraseand thermal denaturation of nicking enzyme were four importantfactors that should be balanced for seeking an optimal incubationtemperature. The optimization of incubation temperature (Fig. S4)shows that 1 nM TS-mutant can trigger significant signal at 58 1Cthat is much better than those performed at 55 1C, 60 1C and 63 1C.We prefer to suppose that although the activity of Nt.BstNBI mightbe slightly restricted compared with that at 55 1C (the optimumtemperature for Nt.BstNBI activity), higher temperature probablyassisted the dissociation of created X (here the TS-mutantsequence) from an X0–X0 template, which immediately took partin another exponential circuit rapidly. The temperature adjust-ment offered us a potential way to improve performance towardsother target oligonucleotide with different length and specific

sequence. At 58 1C, we further optimized the incubation time ofGQ-EXPAR. As a better sensitivity toward TS-mutant was achievedwith longer incubation time (Fig. S5), 1 h was selected as a suitablecondition.

The quantitative performance of GQ-EXPAR for Tay–Sachsmutant sequence was investigated under the optimum conditionsmentioned above. As shown in the corresponding calibrationcurve (Fig. 5), the signal response increased with the concentrationof TS-mutant ranging from 5 pM to 1 nM. And 5 pM TS-mutantwas easily detectable by naked eye. The LOD was estimated to be1.7 pM (17 amol in 10 μL) based on an S/N of 3, which is compar-able or even better than the recently reported assays for Tay–Sachsmutant detection (Lu et al., 2012; Wang et al., 2012). For compar-ison, both 100 pM mutant gene and normal gene were analyzed,individually. A five-fold colorimetric signal for mutant gene wasobserved, implying that the mutant can be discriminated from thenormal gene (Fig. S6).

4. Conclusion

In summary, we developed a novel GQ-EXPAR for simple, rapid,and label-free DNA colorimetry. It was achieved by flexiblyengineering exponential and linear amplification circuits to bringout the continuous creation of trigger DNA and their conversion tosignal readout element, a G-quadruplex sequence EAD2. EXPARintegrated with G-quadruplex/hemin DNAzyme contains followingadvantages: (1) the tandem dual-amplification, exponential circuitfor trigger DNA and mimic-HRP DNAzyme, is involved to guaran-tee good quantification performance; (2) the label-free and one-pot homogeneous DNA detection can be simply finished in an hourwithout any extra synthesis or separation process; (3) reporteroligonucleotide is directly utilized to produce the signal instead ofcomplicated downstream operations. The strategy can detect aslow as 2.5 pM model DNA with high specificity towards mis-matched/perfect-matched sequences. The successful detection ofTay–Sachs mutant (LOD 1.7 pM) illustrated the feasibility of theGQ-EXPAR for DNA detection to be implemented to many moreoligonucleotides. We envision the GQ-EXPAR to be a potential toolfor point-of-care molecular diagnostics.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Nos. 21275009 and 20805002) and theScientific Research Foundation for the Returned Overseas ChineseScholars, MOE, China.

Appendix A. Supplementary material

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bios.2014.01.032.

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