This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 3573--3575 3573

Cite this: Chem. Commun.,2013,49, 3573

Sequence-specific recognition of double-stranded DNAwith molecular beacon with the aid of Ag+ underneutral pH environment†

Zhiyou Xiao, Xiaoting Guo and Liansheng Ling*

A sensitive fluorescent sensor for sequence-specific recognition of

dsDNA was established with a molecular beacon (MB) based upon

the formation of parallel triplex DNA in the presence of Ag+ under

neutral pH environment.

The double-stranded structure of DNA was reported in 1953,which revealed the nature of heredity.1,2 Double-strandedstructure was the natural state of DNA, so the direct recognitionof double-stranded DNA (dsDNA) was of importance in the fieldof disease diagnosis, modulating of gene expression, andgene therapy.3–8 Therefore, sequence-specific recognition of dsDNAhas attracted much attention, and many materials such as poly-amides,9,10 zinc-finger DNA-binding proteins,11,12 and triplex formeroligonucleotides8,13–18 have been applied to recognize dsDNA.

The molecular beacon (MB) was a single-stranded DNA(ssDNA) probe with special stem-loop structure, which was firstproposed by Tyagi and Kramer in 1996.19 Afterwards, the MBwas widely used in the recognition analysis,20 such as detectionof DNA and RNA,19,21–23 study of the interaction between proteinand DNA,24–26 establishment of biosensors,27,28 and fabricationof biochips.27,29,30 Most applications of the MB were based onthe fluorescence enhancement caused by its hybridization withsingle-stranded target DNA (or RNA).

Homopurine�homopyrimidine duplex DNA could be recognizedwith oligonucleotide by forming triplex DNA.8,14,17,18,31,32 Thehomopyrimidine strand could bind to the major groove ofdsDNA with a parallel orientation, and homopurine strandcould bind to the homopurine strand of the dsDNA with ananti-parallel orientation.33 Anti-parallel triplex DNA could beformed under a neutral pH environment. Whereas the cytosineneed to be protonated for forming C�G3C triads (� denotesWatson–Crick bond, 3 denotes Hoogsteen bond) in the type ofparallel triplex DNA so the stability of triplex DNA containing aC�G3C triad depends on the pH value, it was usually formed

under acidic pH environment and could not be formed underphysiological conditions.34,35 Recently, Ihara and co-workersreported that Ag+ could specifically bind to C� G3 C triad, whichmade it could be formed under neutral pH environment.36

Herein we develop a fluorescent method for sequence specificrecognition of dsDNA with MB through triplex formation withthe aid of Ag+ under neutral pH environment. Oligonucleotide50-6-FAM-GTGGAG�T�T�C�T�T�T�C�T�T�C�T�C�T�T�T�C�C�TCTCCAC-BHQ-1-30 isdesigned as the MB. The underlined portion is the loop region,which is designed for the recognition of target dsDNA. Twoends of the loop region are complementary stem portions, thefluorophore (6-FAM) is labeled at the 50 end, and the quencher(BHQ-1) is labeled at the 30 end. All oligonucleotides are listedin Table 1. Target dsDNA (T) is composed of two complementaryoligonucleotides (Ta and Tb), which can hybridize with the loopportion of MB and form triplex DNA through the formation ofC�G3C and T�A3T triads.

The scheme of the assay is shown in Fig. 1, stem-loop structureof the MB brings the fluorophore into the close proximity of thequencher, and ‘‘turn off’’ the fluorescence of the MB. Uponaddition of the target dsDNA and Ag+, hybridization occurs betweentarget dsDNA and loop portion of the MB, induces the formation oftriplex DNA and ‘‘turn on’’ the fluorescence.

To investigate the probability of the scheme, the fluorescencespectrum of the MB was obtained under different conditions.As shown in Fig. 2, the free MB demonstrated weak emission,and there was little change upon addition of Ag+. When targetdsDNA was added into the MB solution, its emission increasedmanifestly, which indicated the formation of triplex DNA and

Table 1 Sequence of oligonucleotides

Name Sequence Abbrev.

Target dsDNA (T) 50-AAGAAAGAAGAGAAAGGA-30 Ta

30-TTCTTTCTTCTCTTTCCT-50 Tb

Control dsDNA-1 (T1) 50-AAGAAAGAA�AAGAAAGGA-30 T1a

30-TTCTTTCTT�TTCTTTCCT-50 T1bControl dsDNA-2 (T2) 50-AAG�GAAGAAGAGAAA�AGA-30 T2a

30-TTC�CTTCTTCTCTTT�TCT-50 T2b

School of Chemistry and Chemical Engineering, Sun Yat-Sen University,

Guangzhou 510275, P. R. China. E-mail: cesllsh@mail.sysu.edu.cn

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc40333e

Received 15th January 2013,Accepted 5th March 2013

DOI: 10.1039/c3cc40333e

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3574 Chem. Commun., 2013, 49, 3573--3575 This journal is c The Royal Society of Chemistry 2013

partial restoration of MB fluorescence, which might be due tothe weak stability of parallel triplex DNA under neutral pHenvironment.31,32,37 However, the fluorescence intensity of MBenhanced dramatically when the MB was mixed with targetdsDNA and Ag+ together, which was in good accordance toreports that Ag+ could recognize the C�G3C triads and enhancethe stability of triplex DNA under a neutral pH environment.36

To explore the mechanism of the fluorescence enhancementof the MB, the circular dichroism spectroscopy of the MB wasinvestigated under different conditions (see ESI†). An obviousnegative cotton effect at 247 nm for target dsDNA was observed,which indicated the helicity of target dsDNA. The mixture of theunlabeled MB and target dsDNA had an obvious negative peakaround 247 nm, a stronger positive peak around 276 nm thatrelevant to base stacking, and a weak negative peak around210 nm, which indicated the formation of some triplex DNA.10

Upon the addition of Ag+, the indicator of triplex DNA (210 nm)and other peaks got much stronger, which suggested that Ag+

could enhance the stability of triplex DNA,36 these were in goodagreement with the result of fluorescence.

Parallel triplex DNA which contain C�G3C triads could beformed only in the acid environment, which is due to thenecessity of protonation of cytosine.31,32 However, Ag+ canspecifically recognize the C�G3C triad and enhance the stabilityof triplex DNA,36 which enables it to form triplex DNA under aneutral pH environment, so the effect of the concentration ofAg+ was studied (see ESI†). The DI increased along with concen-tration of Ag+ over the range from 0.05 to 0.40 mM, which indicated

that Ag+ could enhance the stability of triplex DNA during thisrange, and recover the fluorescence of the MB. However, itdecreased gradually when the concentration of Ag+ varied overthe range of 0.40–1.60 mM, which might be due to the formationof the C–Ag–C complex in the loop portion of the MB in thepresence of excess Ag+.38,39 Therefore, 0.40 mM of Ag+ wasselected for the research.

Under the conditions of 100 nM MB, 0.40 mM of Ag+, 0.3 mMof spermine, pH 7.4 and 90 minutes of hybridization time (seeESI†), the fluorescence intensity increased linearly with theconcentration of target dsDNA over the range from 750 pMto 50 nM. As shown in Fig. 3, the linear regression equation wasDI = 2.38C + 18.8 (C: nM, r2 = 0.997), the detection limit was693 pM (3d/slope). The relative standard derivation (n = 9) for6.0 nM target dsDNA was 2.99%. Although the sensitivity of thesensor was lower than that of the dynamic light scattering (DLS)method,31 they were suitable for different targets; the newsensor was suitable for a target with shorter sequence (onerecognition part, around 18 base pairs), while the target for theDLS sensor should be longer than 30 base pairs, because itneeded two recognition parts. Therefore, the proposed methodwas complementary for the DLS sensor.

Sequence specific recognition of dsDNA based upon theformation of parallel triplex DNA by means of forming C�G3Cand T�A3T triads. To estimate the sequence specificity of theassay, we designed two control dsDNA, which were controldsDNA-1 (T1) and control dsDNA-2 (T2), respectively. As listed

Fig. 1 Scheme of the fluorescent sensor for sequence-specific recognition oftarget dsDNA.

Fig. 2 Fluorescence spectra of the MB under different conditions. 20 mM PBS(pH 7.4), 15.0 mM NaNO3, 0.30 mM spermine; (a) 100 nM MB, (b) a + 0.40 mMAg+, (c) a + 100 nM target dsDNA, (d) c + 0.40 mM Ag+.

Fig. 3 (A) The fluorescence spectrum of the MB in the presence of differentconcentration of target dsDNA; (B) the plot of the assay. 20 mM of PBS (pH 7.4),100 nM of MB, 15.0 mM of NaNO3, 0.30 mM of spermine, 0.40 mM of Ag+,incubation time of 1.5 h, target dsDNA: (1) 0 nM, (2) 0.75 nM, (3) 1.5 nM,(4) 3.0 nM, (5) 6.0 nM, (6) 12.0 nM, (7) 25.0 nM, (8) 50.0 nM, (9) 100.0 nM.

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in Table 1, compared with that of target dsDNA (T), one CGbase pair in T1 was replaced by TA base pair, and two base pairsin T2 were replaced. As shown in Fig. 4, the DI for the targetdsDNA was 115.5, while they were 29.8 and 1.5 for controlT1 and T2, respectively.

Molecular beacon (MB) was a dual-labeled oligonucleotideprobe with special stem-loop structures, which has no emissionin the absence of target molecule. Here we constructed asequence-specific fluorescent sensor for dsDNA by using MB witha sequence of 50-6-FAM-GTGGAGTTCTTTCTTCTCTTTCCTCTC-CAC-BHQ-1-30, it had no fluorescence because of fluorescenceresonance energy transfer, and its fluorescence was ‘‘turned on’’upon the addition of target dsDNA. Under the optimized condi-tions, the fluorescence intensity was proportional to the concen-tration of target dsDNA over the range from 750 pM to 50 nM.Meanwhile the recognition of target dsDNA could be accom-plished under the neutral pH conditions with the aid of Ag+,which breakthrough the limit that parallel triplex DNA whichcontain C� G3 C triads could be formed only in the acidenvironment. Moreover, the assay had the merit of highlysequence-specific recognition property, which enabled it apowerful method for solving the problem that derived from thecontaminated DNA during the PCR process in the future.

This work was supported by National Natural SciencesFoundation of China (NSFC No: 20975116)

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Fig. 4 The selectivity of the assay. The experiment was performed under thefollowing conditions: 20 mM of PBS (pH 7.4), 100 nM of MB, 15.0 mM NaNO3,0.30 mM of spermine, 0.40 mM of Ag+, 50 nM of target dsDNA (T) or control DNA(T1 or T2), incubation time of 1.5 h.

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