Multiplexed Analysis of Genes and of Metal Ions Using Enzyme

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Multiplexed Analysis of Genes and of Metal Ions Using Enzyme/DNAzyme Amplification Machineries Lina Freage, Fuan Wang, Ron Orbach, and Itamar Willner* Institute of Chemistry, The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *

ABSTRACT: The progressive development of amplified DNA sensors using nucleic acid-based machineries, involving the isothermal autonomous synthesis of the Mg2+-dependent DNAzyme, is used for the amplified, multiplexed analysis of genes (Smallpox, TP53) and metal ions (Ag+, Hg2+). The DNA sensing machineries are based on the assembly of two sensing modules consisting of two nucleic acid scaffolds that include recognition sites for the two genes and replication tracks that yield the nicking domains for Nt.BbvCI and two different Mg2+-dependent DNAzyme sequences. In the presence of any of the genes or the genes together, their binding to the respective recognition sequences triggers the nicking/polymerization machineries, leading to the synthesis of two different Mg2+-dependent DNAzyme sequences. The cleavage of two different fluorophore/quencher-modified substrates by the respective DNAzymes leads to the fluorescence of F1 and/or F2 as readout signals for the detection of the genes. The detection limits for analyzing the Smallpox and TP53 genes correspond to 0.1 nM. Similarly, two different nucleic acid scaffolds that include Ag+-ions or Hg2+-ions recognition sequences and the replication tracks that yield the Nt.BbvCI nicking domains and the respective Mg2+-dependent DNAzyme sequences are implemented as nicking/replication machineries for the amplified, multiplexed analysis of the two ions, with detection limits corresponding to 1 nM. The ions sensing modules reveal selectivities dominated by the respective recognition sequences associated with the scaffolds.

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recognition-probe/DNA-analyte duplexes or of the recognition aptamer−substrate complexes and the regeneration of the respective analytes.17 Besides the need to develop DNA machineries for the highly sensitive detection of the analytes, the development of multiplexed DNA sensing machineries, for the parallel detection of several analytes, is a continuous analytical challenge. Different approaches for the multiplexed analysis of DNAs,18 aptamer− substrate complexes,19 or metal ions20 were reported. For example, different sized CdSe/ZnS quantum dots functionalized with gene-specific probes modified with quencher units were implemented for the amplified multiplexed optical detection of genes using the exonuclease III-mediated regeneration of the analyte DNAs.18a Similarly, the selective desorption of probes, modified with different fluorophores, from graphene oxide supports by the analyte genes and the regeneration of the analytes by the exonuclease III digestion of the desorbed duplex structures were used for the amplified multiplexed fluorescence detection of genes.18b Also, the multiplexed analysis of metal ions was reported using different sized quantum dots modified with ion-specific binding nucleic acids.20a Here, we report on the development of versatile sensing platforms for the amplified and

he development of amplified detection platforms that implement nucleic acids as recognition elements for analyzing genes, aptamer−substrate complexes, or metal ions attracted substantial research efforts in the past two decades.1,2 Numerous studies have used enzymes3 or metallic nanoparticles as electrocatalytic labels for amplifying the sensing events, and plasmonic metal nanoparticles4 were applied for the amplification of recognition events.5 Furthermore, extensive efforts are recently directed to develop enzyme machineries,6 all-nucleic acid catalytic DNA machineries,7 and composite enzyme/ DNAzyme machineries8 for the isothermal amplified detection of DNA,9 aptamer−substrate complexes,10 or metal ions.11 For example, all-nucleic acid machineries for the detection of DNA have used the hybridization chain reaction (HCR) as an isothermal autonomous process for the synthesis of catalytic nucleic acid (DNAzymes, such as the Mg2+-dependent RNAcleavage DNAzyme) wires.12 Enzyme/DNAzyme coupled amplification machineries have involved the isothermal rolling circle amplification (RCA) process that synthesized the hemin/ G-quadruplex DNAzyme wires for the colorimetric or chemiluminescence detection of DNA.13 A versatile enzyme/ DNAzyme machinery for the detection of DNA,14 aptamer− substrate complexes,15 or Hg2+ ions16 has involved an isothermal autonomous replication/nicking process that synthesizes DNAzymes as catalytic probes for the optical detection of the analytes. All-enzyme-based amplification machineries have involved the exonuclease III, Exo III, cleavage of the © 2014 American Chemical Society

Received: August 17, 2014 Accepted: October 20, 2014 Published: October 20, 2014 11326

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Table 1. DNA Sequences Used to Construct the Sensing Platforms No.

sequence

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

5′-CGTATTTCATGGTATA-3′ 5′-TAGGATAACATGGGTGTAACCTGGTTAATCGCTGAGTACTGCTGAGGTATACCATGAAATACG-3′ 5′-AGTACTAGCGATTAACCAGGTTACACCCATGTTATCCTA-3′ 5′-FAM-TAGGATATrAGGAGTACT-Iowa Black RQ-3′ 5′-TCGTAGGATTGATCTAAACT-3′ 5′-TGACTGTACATGGGTGTAACCTGGTTAATCGCTGAATGACGCTGAGGAGTTTAGATCAATCC-3′ 5′-GTCATTCAGCGATTAACCAGGTTACACCCATGTACAGTCA-3′ 5′-ROX-TGACTGTTrAGGAATGAC-BHQ2-3′ 5′-TAGGATAACATGGGTGTAACCTGGTTAATCGCTGAGTACTGCTGAGGCAATGGAAAAAACCATTC-3′ 5′-TGACTGTACATGGGTGTAACCTGGTTAATCGCTGAATGACGCTGAGGTAATGGAAAAAACCATTT-3′

multiplexed analysis of genes or of metal ions (Hg2+ or Ag+) using autonomous polymerization machineries that operate on DNA scaffolds. The machineries lead to the generation of sequence-specific Mg2+-dependent DNAzymes that yield to different fluorescence signals dictated by the respective analytes.

50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol. For the detection of the Ag+ ions, the working solution included 0.5 μM DNA template (9), 0.25 μM DNA strand (3), and 0.75 μM of the DNAzyme substrate (4). The solution was annealed by warming the solution to 95 °C for 5 min and subsequently cooling to 30 °C for 2 h, followed by the addition of dNTPs (0.5 mM), Klenow fragment (0.2 U/μL), Nt.BbvCI (0.2 U/μL), and different concentrations of the Ag+ ions to the mixture. For the detection of the Hg2+ ions, the working solution included 0.5 μM DNA template (10), 0.25 μM DNA strand (7), and 0.75 μM of the DNAzyme substrate (8). The solution was annealed by warming the solution to 95 °C for 5 min and subsequently cooling to 30 °C for 2 h. dNTPs (0.5 mM), Klenow fragment (0.2 U/μL), Nt.BbvCI (0.2 U/μL), and different concentrations of the Hg2+ ions were added to the mixture. For the multiplexed detection of the two different metal ions, Ag+ and Hg2+, the working solution included 0.5 μM DNA templates (9) and (10), 0.25 μM DNA strands (3) and (7), and 0.75 μM of the DNAzyme substrates (4) and (8). The solution was annealed by warming the solution to 95 °C for 5 min and subsequently cooling to 30 °C for 2 h. Then, dNTPs (0.5 mM), Klenow fragment (0.2 U/μL), Nt.BbvCI (0.2 U/μL), and different metal ions, Ag+ and Hg2, were added to the mixture. The resulting solution (150 μL) was transferred to a cuvette, and the timedependent fluorescence changes were monitored spectroscopically at 30 °C. The fluorescence spectra corresponding to each of systems were recorded after a fixed time interval specified for each of the systems.

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EXPERIMENTAL SECTION Materials. Klenow fragment (3 → 5 exo-), dNTPs, and Nt.BbvCI nicking enzyme were purchased from New England Biolabs Inc. (Beverly, MA, USA). All DNA oligonucleotides were purchased from Sigma. Table 1 shows the sequences of the oligonucleotides used in the study. The oligonucleotides were HPLC-purified and dissolved in water to yield stock solutions of 100 μM. Ultrapure water from a NANOpure Diamond (Barnstead) source was used in all of the experiments. Detection of the DNA by Using the Polymerization/ Nicking DNA Machineries. All the assays were prepared in 1× NEBuffer 2 buffer that contained 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol. For the detection of the target Smallpox gene (1), the working solution included 0.5 μM DNA template (2), 0.25 μM DNA strand (3), and 0.75 μM of the DNAzyme substrate (4). The solution was annealed by warming the solution to 95 °C for 5 min and subsequently cooling to 30 °C for 2 h. Next, dNTPs (0.5 mM), Klenow fragment (30 U/μL), Nt.BbvCI (0.2 U/μL), and different concentrations of the target gene (1) were added to the mixture. For the detection of the target TP53 gene (5), the working solution included 0.5 μM DNA template (6), 0.25 μM DNA strand (7), and 0.75 μM of the DNAzyme substrate (8). The solution was annealed by warming the solution to 95 °C for 5 min and subsequently cooling to 30 °C for 2 h, followed by the addition of dNTPs (0.5 mM), Klenow fragment (0.2 U/μL), Nt.BbvCI (0.2 U/μL), and different concentrations of the target gene (5) to the mixture. For the multiplexed detection of the Smallpox (1) and TP53 (5) genes, the working solution included 0.5 μM DNA templates (2) and (6), 0.25 μM DNA strand (3) and (7), and 0.75 μM of the DNAzyme substrates (4) and (8). The solution was annealed by warming the solution to 95 °C for 5 min and subsequently cooling to 30 °C for 2 h. Next, dNTPs (0.5 mM), Klenow fragment (0.2 U/μL), Nt.BbvCI (0.2 U/μL), and different target genes (1) or/and (5) were added to the mixture. The resulting solution (150 μL) was transferred to a cuvette, and the time-dependent fluorescence changes were monitored spectroscopically at 30 °C. The fluorescence spectra corresponding to each of the systems were recorded after a fixed time interval specified for each of the systems. Detection of the Metal Ions by Using the Polymerization/ Nicking DNA Machineries. All assays were prepared in 1× NEBuffer 2 buffer that contained 10 mM Tris-HCl (pH 7.9),

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RESULTS AND DISCUSSION The multiplexed analysis of two different genes (Smallpox virus gene and TP53 gene) is schematically described in Figure 1A. The sensing module for analyzing Smallpox gene fragment (1) consists of a predesigned scaffold (2) that is hybridized with a blocker unit (3). The scaffold (2) is composed of three domains, I, II, and III, as depicted in Figure 1A, panel A. Domain I corresponds to the gene recognition sequence, and it is complementary to the sequence I′ associated with target gene (1). Domain III of the scaffold (2) is complementary to the Mg2+dependent DNAzyme sequence. Domain II of the scaffold (2) includes a sequence-specific domain that, upon formation of a duplex structure with the complementary strand II′, yields the nicking domain to be cleaved by the nicking enzyme Nt.BbvCI. The blocker unit (3) hybridized with the scaffold (2) consists of the sequence III′ (the Mg2+-dependent DNAzyme sequence) and a tether that is partially complementary to the nicking domain II. The hybrid between the scaffold (2) and the blocker unit (3) provides the sensing module. In the presence of the analyte gene (1), the nucleotide mixture dNTPs, the polymerase 11327

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Figure 1. (A) Scheme corresponding to polymerization/nicking DNA machineries yielding the Mg2+-dependent DNAzymes as fluorescence transduction biocatalysts for the analysis and multiplexed analysis of the Smallpox (1) and TP53 (5) genes. (B) Time-dependent fluorescence changes upon analyzing the Smallpox gene (1) according to the machinery shown in (A), panel A. (C) Fluorescence spectra corresponding to the analysis of variable concentrations of target gene (1), for fixed time-interval of 12 h, according to (A), panel A: (a) 0, (b) 0.1, (c) 0.5, (d) 1.0, (e) 2.0, (f) 5.0, (g) 10, (h) 20, (i) 50, and (j) 100 nM. Inset: Derived calibration curve corresponding to the fluorescence changes generated by different concentrations of the Smallpox gene (1). Experimental conditions are outlined in the Experimental Section.

of the substrate, the formation of the complex (3)/(4) and the subsequent cleavage of the substrate (4) lead to the fragmentation of the substrate and to the turned “ON” fluorescence of F1, that provides the readout signal for the sensing event. Note that the sensing machinery could be activated and lead to the DNAzyme sequence, even in the absence of the blocker unit. Nonetheless, the blocker unit (3), being a part of the sensing module, has an important function in the sensing platform. The displaced strand (3) is complementary to the scaffold. Hence, by using an unblocked scaffold as sensing module would introduce a competitive process, where the association of the released strand (3) to unblocked scaffolds competes with the self-assembly of the Mg2+-dependent DNAzyme that provides the readout signal. Blockage of the scaffolds by (3) prohibits the undesired hybridization of (3) with the scaffold, thus enabling the full utilization of the displaced strand as a catalytic DNAzyme reporter. Note that the Mg2+-dependent DNAzyme includes a conserved loop sequence extended at its 3′- and 5′-ends with nonconserved sequences Ni and Nj, and these provide the

enzyme, and the nicking enzyme, the autonomous replication/ nicking machinery is activated. That is, the analyte gene (1) binds to the recognition domain I and initiates the replication of the scaffold while displacing the blocker unit (3). Replication of the scaffold (2) yields the duplex domain II/II′ that provides the nicking site in II′. The resulting nicked site provides an opening for polymerase, leading to the rereplication of the scaffold and the displacement of the strand (3) formed in the former replication cycle. Namely, the association of the gene (1) to the recognition site initiates the isothermal autonomous continuous replication of the scaffold (2) and the release of the sequence (3). The released strand (3) includes, however, the base sequence that folds into the catalytically active loop structure of the Mg2+dependent DNAzyme. As the DNAzyme substrate (4) is modified by the fluorophore/quencher pair F1/Q1 (F1 = FAM, Q1 = Iowa Black RQ), paired as an auxiliary component, the formation of the catalytically active DNAzyme loop structure (3) leads to the assembly of the DNAzyme/substrate structure (3)/(4). While the fluorophore F1 is quenched in the intact structure 11328

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binding sites for the DNAzyme substrates. As Ni and Nj can be altered, different DNAzyme substrate strands, modified by different fluorophore/quencher pairs, can be engineered. For the analysis of target gene (1), the displaced DNAzyme strand (3) includes the domains Ni = N1 and Nj = N2, and the substrate (4) includes the complementary sequence. The sensing of the second TP53 gene (5) implements the same paradigms, yet it requires the reprogramming of the scaffold machinery and the use of a different fluorophore/quencher-functionalized substrate for the displaced DNAzyme, Figure 1A, panel B. For sensing the TP53 gene fragment (5), a different scaffold (6) is programmed, and it consists of the recognition domain IV, the sequence II that yields in the presence of the complementary domain II′ the nicking site and domain V that is composed of a region complementary to the conserved region of the Mg2+-dependent DNAzyme extended by nonconserved domains Ni = N3 and Nj = N4. The substrate for the Mg2+-dependent DNAzyme generated by the sensing module of gene (5) is substituted with fluorophore/quencher pair F2/Q2 (F2 = ROX, Q2 = BHQ2). The scaffold (6) is blocked by (7). The substrate (8) includes the sequence complementary to the engineered DNAzyme, and it includes the conserved sequences and the sequence-specific complementary tethers N3′ and N4′. The recognition of gene (5), by the sensing module (6)/(7), in the presence of the enzymes polymerase, the nicking enzyme, the nucleotide mixture dNTPs, and the auxiliary substrate (7), activates the autonomous sensing machinery. Binding of the gene TP53 (5) to the sensing module (6)/(7) initiates the replication/nicking machinery that leads to the displacement of the DNAzyme sequence (7). The displaced DNAzyme binds to the substrate (8), leading to the cleavage of (8) and to the release of the F2-modified fragmented substrate. The fluorescence of F2 provides then the readout signal for the detection of gene TP53 (5). Figure 1B depicts the time-dependent fluorescence spectra upon analyzing a fixed concentration (100 nM) of the Smallpox gene (1), according to Figure 1A. The fluorescence spectra intensify with time, consistent with the continuous cleavage of DNAzyme substrate (4) as the reaction proceeds. The fluorescence changes of the system tend to level off to a saturation value after ca. 12 h, due to the depletion of the dNTPs, and the partial deactivation of the nicking enzyme. Accordingly, different concentrations of the gene (1) were analyzed by the sensing machinery shown in Figure 1A, panel A, using a fixed timeinterval of 12 h. Figure 1C depicts the fluorescence spectra changes upon analyzing different concentrations of (1) for a fixed time-interval of 12 h. The resulting calibration curve is shown in Figure 1C, inset. The system enabled the detection of the gene (1) with a detection limit corresponding to 0.1 nM. Similarly, the sensing module shown in Figure 1A, panel B, was applied for the sensing of gene TP53 (5). The experimental results are depicted in Figure S1, Supporting Information. The system enabled the analysis of gene (5), with a detection limit corresponding to 0.1 nM. The significance of the blocker unit (3) in enhancing the amplified detection of the Smallpox gene (1) is demonstrated in Figure 2. We compared the time-dependent fluorescence changes generated by the machinery shown in Figure 1A, panel A, in the presence of the blocker, upon analyzing the gene (1) at concentrations corresponding to 100 nM, curve (a), and 50 nM, curve (b), to the fluorescence changes generated by the machinery that lacks the blocker unit, upon analyzing 100 and 50 nM of gene (1), curve (a′) and (b′), respectively. Evidently, the fluorescence changes are substantially higher and faster, in the presence of the blocker machinery. For example, while the

Figure 2. Time-dependent fluorescence changes upon analyzing (a) 100 and (b) 50 nM of the Smallpox gene (1), using the blocked sensing machinery shown in Figure 1A, panel A, and upon analyzing (a′) 100 and (b′) 50 nM of the gene (1) by an unblocked machinery.

fluorescence changes of the unblocked machinery, upon analyzing 50 nM of gene (1) are almost negligible, the fluorescence changes generated by the blocked machinery upon analyzing 50 nM (1) are high; see curves (b′) vs (b). The lower performance of the unblocked sensing machinery is attributed to the preferred rehybridization of the displaced DNAzyme strand (3) to the track (2) (particularly at low concentrations of (3)). Similar effects of the blocker unit (V′) on the enhanced amplified detection of the TP53 gene, by the blocked machinery shown in Figure 1A, panel B, are observed upon comparing the system to the unblocked machinery, Figure S2, Supporting Information. The successful analysis of the two genes, (1) and (5), by the sensing modules shown in Figure 1A, using the fluorophores F1 and F2 as readout signals, enables then the multiplexed, parallel, analysis of the two different genes. Accordingly, the two sensing modules shown in Figure 1A and the composite system were subjected to the individual genes or to a mixture of the two genes. Figure 3A shows that no fluorescence changes occur in the composite of sensing modules in the absence of target genes (1) or (5). Treatment of the mixture of sensing modules with the Smallpox gene (1) or the TP53 gene (5) turns on the fluorescence of F1 or F2, respectively, Figure 3B,C. Subjecting the mixture of sensing modules to the two genes (1) and (5) triggers on the fluorescence of F1 and F2, Figure 3D, demonstrating the multiplexed analysis of the two genes. (For the time-dependent fluorescence changes upon multiplexed analysis of the different target DNAs, see Figure S3, Supporting Information.). The replication/nicking machinery that autonomously synthesizes the Mg2+-dependent DNAzyme that leads to a fluorescence readout signal was further applied for the amplified and multiplexed analysis of Ag+ ions and Hg2+ ions, Figure 4A. The metal ions are sensed using two sensing modules, T1 and T2, for the detection of Ag+ and Hg2+ ions, respectively. The sensing module, T1, for analyzing Ag+ ions is composed of the scaffold (9) and the blocker unit (3), Figure 4A, panel A. The scaffold (9) consists of three domains, where domain VI includes cytosine (C) bases and provides the recognition sequence for Ag+. The domain II includes the base sequence that upon replication yields the duplex II/II′, where II′ yields the nicking site for Nt.BbvCI. Domain III in the scaffold includes the complementary sequence of the Mg2+-dependent DNAzyme that includes the sequences for hybridization of the substrate (4). The blocker unit (3) hybridized with the scaffold (9) includes the Mg2+-dependent DNAzyme sequence, and this is extended by domains N1 and N2. The substrate (4) includes the conserved ribonucleobase sequence characteristic for the Mg2+-dependent DNAzyme, 11329

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the hybrid (3)/(9), was demonstrated by comparing the performance of the sensing module T1 to the unblocked machinery composed of the scaffold (9) only. Figure S5, Supporting Information, shows the time-dependent fluorescence changes upon analyzing different concentrations of Ag+ ions in the presence of the unblocked system consisting of the scaffold (9) only. These results should be compared to the time-dependent fluorescence changes generated by the sensing module T1, Figure S4, Supporting Information. Evidently, the fluorescence changes are substantially higher in the presence of the blocked sensing module T1. In fact, the sensing module T1 enabled the detection of Ag+ at concentrations that could not be detected by the unblocked scaffold (9). (For example, T1 enabled the sensing of Ag+ in the range of 1−5 nM while the unblocked scaffold (9) did not show any fluorescence changes at Ag+, 5 nM.) Figure 5 depicts the calibration curve corresponding to the fluorescence changes generated upon analyzing different concentrations of Ag+ by the blocked sensing module T1, curve (a), and by the unblocked scaffold, curve (b). Evidently, the blocked scaffold T1 shows substantial fluorescence changes at low concentrations of Ag+ ions (1−5 nM region). The limited performance of the unblocked scaffold (9) toward sensing of Ag+ ions is attributed to the preferred rehybridization of the displaced DNAzyme (3) to the scaffold (particularly at low concentrations of the DNAzyme generated by low concentrations of Ag+ ions). A similar concept was applied for the amplified sensing of Hg2+ ions, Figure 4A, panel B. The sensing module consists of the scaffold (10) that includes three functional domains VII, II, and V. The domain VII acts as the Hg2+-ions recognition site and includes thymine (T) base as ligation sites for Hg2+ ions. Domain II includes the base sequence that upon replication yields a duplex structure with the instructive base sequence to be nicked by Nt.BbvCI. Domain V includes the base sequence that is complementary to the Mg2+-dependent DNAzyme sequence generated by the replication/nicking machinery. The scaffold (10) is blocked by the strand (7). The nucleic acid (8) is, also, introduced into the system, and it acts as the substrate for the Mg2+-dependent DNAzyme generated by the sensing machinery. The substrate (8) is substituted by a fluorophore/quencher pair F2/Q2 (F2 = ROX, Q2 = BHQ2), leading to the quenching of the fluorophore. The DNAzyme sequence generated by the machinery includes the characteristic Mg2+-dependent DNAzyme loop that is extended, by the sequences N3 and N4 that hybridized with domains N3′ and N4′ linked to the substrate. The substrate (8) consists of the conserved ribonucleobase sequence that is extended by domains N3′ and N4′. This results in the specific sequence of the Mg2+-dependent DNAzyme being synthesized by the sensing machinery. In the presence of Hg2+ ions, the recognition sequence folds into a hairpin structure that is stabilized by bridging thymine−Hg2+−thymine (T−Hg2+−T) bridges on the scaffold (10). The resulting hairpin generated on the scaffold triggers in the presence of polymerase, dNTPs, and the nicking enzyme Nt.BbvCI, the replication of the scaffold, and the displacement of the blocker unit (7). The replicated strand includes the nicking site, and the nicked strands activate the autonomous polymerization/nicking machinery accompanied by the displacement of (7) that consists of the Mg2+-dependent DNAzyme sequence that binds the substrate (8). The DNAzyme-induced cleavage of the substrate (8) results in the fragmented fluorophore-functionalized strand, and the fluorescence generated by F2 provides a quantitative readout signal for the Hg2+ ions. Figure S6, Supporting Information, depicts the time-dependent fluorescence spectra changes of F2 upon sensing

Figure 3. Multiplexed analysis of the Smallpox (1) and TP53 (5) genes using the two sensing modules depicted in Figure 1A. (A) The fluorescence changes of the two sensing modules in the absence of the genes. (B) Fluorescence changes upon subjecting the two sensing modules to gene (1). (C) Fluorescence changes upon subjecting the two sensing modules to gene (5). (D) Fluorescence changes upon subjecting the two sensing modules to (1) and (5). In all experiments, the concentrations of (1) and (5) correspond to 10 nM. (a) The fluorescence generated by Smallpox gene (1). (b) The fluorescence generated by the TP53 gene (5). Experimental conditions are outlined in the Experimental Section.

and this is extended at the 3′ and 5′ ends with domains N1′ and N2′ complementary to the region associated with the DNAzyme. The substrate (4) is modified at its ends with the fluorophore/ quencher pair, F1/Q1 (F1 = FAM, Q1= Iowa Black RQ), resulting in the quenching of the fluorophore. In the presence of Ag+ ions, dNTPs, polymerase, and the nicking enzyme Nt.BbvCI, domain VI of the scaffold (9) folds into a hairpin structure stabilized by cytosine−Ag+−cytosine (C−Ag+−C) bridges. This initiates the autonomous replication/nicking machinery, where the replication process displaces the blocker (3), and the subsequent nicking of the replicated product leads to the continuous formation of the Mg2+-dependent DNAzyme. In the presence of Mg2+ ions, the hydrolytic cleavage of the substrate (4) proceeds, and the fragmented fluorophore-functionalized substrate provides the readout signal for the sensing process. Figure S4, Supporting Information, depicts the time-dependent fluorescence changes upon the analysis of Ag+ ions at different concentrations. The fluorescence generated by the system intensifies with time, consistent with the increase of the Mg2+-dependent DNAzyme number of units, and the fluorescence level tends to reach a saturation value after ca. 12 h. Figure 4B depicts the fluorescence spectra generated by the sensing module upon analyzing different concentrations of Ag+ for a fixed time interval of 12 h according to Figure 4A, panel A. As the concentration of Ag+ increases, the fluorescence changes are intensified, consistent with the higher content of the DNAzyme generated as the concentration of Ag+ is elevated, Figure 4B. The resulting calibration curve for analyzing Ag+ ions is shown in Figure 4B, inset. The system enabled the analysis of Ag+ ions with a detection limit that corresponds to 1 nM. The significance of the blocker unit (3) on the enhanced amplified sensing of Ag+ by the sensing module T1, consisting of 11330

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Figure 4. (A) Scheme corresponding to the polymerization/nicking DNA machineries yielding the Mg2+-dependent DNAzymes as fluorescence transduction biocatalysts for the analysis and multiplexed analysis of Ag+ ions and Hg2+ ions: Panel A, Ag+ ions sensing module. Panel B, Hg2+-ions sensing module. (B) Fluorescence spectra changes upon analyzing different concentrations of Ag+ ions, for a fixed time interval of 12 h, by the sensing module shown in (A), panel A: (a) 0, (b) 1, (c) 2, (d) 5, (e) 10, (f) 20, (g) 50, and (h) 100 nM. Inset: Derived calibration curve. (C) Fluorescence spectra changes upon analyzing different concentrations of Hg2+ ions, for a fixed time interval of 12 h, by the sensing module shown in (A) panel B: (a) 0, (b) 1, (c) 2, (d) 5, (e) 10, (f) 20, (g) 50, and (h) 100 nM. Inset: Derived calibration curve. (D) Selectivity pattern upon subjecting the sensing module for detection of Ag+ ions shown in (A), panel A, to different metal ions, 10 nM each. (E) Selectivity pattern upon subjecting the sensing module for detection of Hg2+ ions shown in (A), panel B to different metal ions, 10 nM each. 11331

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fluorescence change only in the presence of Ag+ ions. Similarly, Figure 4E shows the fluorescence intensities generated by the Hg2+-ions sensing module upon its subjection to different ions, in the presence of dNTPs, the enzymes, and the substrate (8), according to Figure 4A, panel B. The system leads to a fluorescence change only in the presence of Hg2+ ions. Evidently, only the ions that are recognized by the recognition sequences of the sensing machineries lead to fluorescence outputs. The successful analysis of the two ions, Ag+ and Hg2+, by the two sensing modules was then applied for the multiplexed, parallel, analysis of the two ions. Accordingly, the two sensing modules shown in Figure 4A were mixed, and the mixture was subjected to the two ions, Figure 6. In the absence of added ions, no fluorescence changes are generated by the system, Figure 6A. In the presence of Ag+ ions, only the Ag+-sensing module is triggered-on while the Hg2+-sensing module is unaffected, giving rise to the fluorescence of only F1, Figure 6B. Similarly, in the presence of the Hg2+-sensing module, the machinery is activated leading to fluorescence changes of only F2, Figure 6C. In the presence of the two ions, Ag+ and Hg2+, the two machineries are simultaneously triggered-on leading to the fluorescence of F1 and F2, Figure 6D. These results reveal the successful parallel analysis of the two ions by the mixture of the two sensing modules. (For the time-dependent fluorescence changes upon multiplexed analysis of the different metal ions, see Figure S7, Supporting Information.)

Figure 5. Calibration curve corresponding to the fluorescence changes in the presence of variable concentrations of Ag+, by (a) the blocked sensing module T1 and (b) the unblocked scaffold (9). Fluorescence changes were measured in the different systems after a fixed timeinterval of 12 h.

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CONCLUSIONS The present study has implemented functional DNA sensing modules based on polymerization/nicking machineries that synthesize the Mg2+-dependent DNAzymes that act as catalytic labels for the sensing processes through the generation of fluorescence readout signals by the fluorophore-modified fragmented substrates of the DNAzymes. The DNA machineries were applied for the detection of Smallpox and TP53 genes and of Hg2+ ions or Ag+ ions. Also, by the mixing of the DNA sensing machineries, the multiplexed, parallel, analyses of the two genes or of the two metal ions were demonstrated. This paradigm can be further extended by applying additional metal-ion-dependent DNAzymes and other fluorophore-functionalized substrates for the analysis of mixtures of genes of enhanced complexities. Also, the incorporation of synthetic nucleic acids into DNA allowed the bridging of duplex DNA structure by the ligation of the nonnative oligonucleotides to transition metal ions. This paves the way to construct a variety of sensing modules for metal ions by the integration of non-native bases into the recognition sequences of the sensing modules.

Figure 6. Multiplexed analysis of Ag+ and Hg2+ ions using the mixture of two sensing modules depicted in Figure 4A. (A) Fluorescence spectra changes of the system without added ions. (B) Fluorescence spectra changes in the presence of only Ag+ ions. (C) Fluorescence spectra changes in the presence of only Hg2+ ions. (D) Fluorescence spectra changes upon subjecting the system to the two ions, Ag+ and Hg2+. The concentration of Ag+ and Hg2+ ions corresponded to 10 nM. (a) The fluorescence generated by Ag+ ions. (b) The fluorescence generated by Hg2+ ions.

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of a fixed concentration of Hg2+ ions. The fluorescence is intensified with time, consistent with the time-dependent increase of the displaced DNAzyme units. Figure 4C depicts the fluorescence spectra changes observed upon analyzing different concentrations of Hg2+ ions according to Figure 4A, panel B. Figure 4C, inset shows the derived calibration curve. The system enabled the analysis of Hg2+ ions with a detection limit that corresponds to 1 nM. The sensing modules shown in Figure 4A for the analysis of Ag+ ions and of Hg2+ ions reveal high selectivity toward the respective ions. Figure 4D shows the fluorescence intensity changes generated upon subjecting the Ag+-ions sensing module to different ions in the presence of dNTPs, the respective enzymes, and the substrate (4), according to Figure 4A, panel A. Evidently, the system generates a

ASSOCIATED CONTENT

S Supporting Information *

Time-dependent fluorescence changes upon analyzing the TP53 gene (5) or Ag+/Hg2+ ions, fluorescence spectra upon analyzing variable concentrations of the TP53 gene (5), and kinetic analysis of the multiplexed detection of the Smallpox virus gene (1) and/or the TP53 gene (5), Ag+, and/or Hg2+ ions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 972-2-6585272. Fax: 972-2-6527715. 11332

dx.doi.org/10.1021/ac5030667 | Anal. Chem. 2014, 86, 11326−11333

Analytical Chemistry

Article

Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by the Israel Science Foundation (Grant No. 1083/12).

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dx.doi.org/10.1021/ac5030667 | Anal. Chem. 2014, 86, 11326−11333