Article pubs.acs.org/ac
Target-Catalyzed Dynamic Assembly-Based Pyrene Excimer Switching for Enzyme-Free Nucleic Acid Amplified Detection Zhihe Qing, Xiaoxiao He,* Jin Huang, Kemin Wang,* Zhen Zou, Taiping Qing, Zhengui Mao, Hui Shi, and Dinggeng He State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, People’s Republic of China ABSTRACT: Because of the intrinsic importance of nucleic acid as biotargets, the simple and sensitive detection of nucleic acid is very essential for biological studies and medical diagnostics. Herein, a new strategy for enzyme-free nucleic acid amplified detection has been opened up by combining the signal-amplification capability of target-catalyzed dynamic assembly with the spatially sensitive fluorescent signal of the pyrene excimer. In this strategy, three metastable pyrenelabeled hairpin DNA probes were designed as assembly components, which were kinetically handicapped from cross-opening in the absence of the target DNA. However, in the presence of the target, the dynamic assembly of branched junctions was circularly catalyzed and accompanied by the switching of the pyrene excimer which emits at ∼488 nm. Thus, the target DNA could be detected by this simple mix-and-detect amplification method, without expensive and perishable protein enzymes. A good detection capability exhibited with a detectable minimum target concentration of 10 pM, which was comparable to or even better than some reported enzyme-dependent amplification methods, and the potential for the target detection from complex fluids was verified. In addition, as a novel transformation of dynamic DNA assembly technology into enzyme-free signal-amplification analytical application, we infer that the proposed strategy will hold promising potential for application in a wider range of fields, including aptamer-based non-nucleic acid target sensing, biomedicine, and bioimaging.
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amplification methods for sensitive nucleic acid analysis.7 The self-assembly based amplification has obvious advantages, compared to conventional enzyme-based methods.8 First, the former is inherently scalable and modular, requiring only the design of base-pairing between metastable DNA “fuel” molecules. Second, the former relies only on hybridization and strand displacement reactions, without requiring perishable protein enzymes, complicated instrumentations and even complex thermal-cycling procedures. However, to the best of our knowledge, the diversity of reported self-assembly-based amplification strategies is very limited and almost all based on HCR4−6 or entropy-driven catalysis.3c,9 Herein, a new enzyme-free nucleic acid amplified detection strategy has been developed by combining the isothermal signal-amplification capability of target-catalyzed dynamic assembly with the spatially sensitive fluorescent signal of pyrene excimers. As detailedly illustrated in Scheme 1, three metastable hairpin DNA probes (A, B, and C) were first designed as “fuel” molecules, a DNA segment was then used as a proof-of-concept nucleic acid target. All DNA sequence information is provided in Table 1. Each hairpin probe was
elf-assembly technology is one of the most important approaches for “bottom-up” fabrication of nanostructures and nanodevices.1 Because of their exquisite recognition properties encoded in the specific interaction of Watson− Crick base-pairing, deoxyribonucleic acids (DNAs) can be programmed and assemble with each other in a predictable manner, which has made DNA the ideal building block for DNA nanotechnology.2 Particularly, the prospect of dynamic DNA self-assembly has vigorously attracted the interest of researchers, and immense advances in this field have been achieved.3 A key feature of the dynamic DNA self-assembly is that it is fueled by the free energy of hybridization of metastable DNA “fuel” molecules and operated in an autonomous and reconfigurable manner. In recent years, inspired by the simple rules governing hybridization of metastable DNA “fuel” molecules, the dynamic DNA self-assembly has provided an attractive signal-amplification approach for biochemical analytical applications.4−7 For example, based on the hybridization chain reaction (HCR),3a in which two metastable DNA hairpin monomers coexist in solution and assemble only upon exposure to an initiator nucleic acid fragment, the Pierce group constructed in situ amplifiers for multiplexed imaging of mRNAs in fixed wholemount and sectioned zebrafish embryos,5 the Tan group built different nanodevices for cancer imaging and therapy,6 and our group has successfully developed fluorescent and colorimetric © 2014 American Chemical Society
Received: January 22, 2014 Accepted: April 21, 2014 Published: April 21, 2014 4934
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formation of the pyrene excimer of aromatic hydrocarbons is restricted to a parallel, aspectant configuration with an interplanar distance of 0.3−0.4 nm.10a However, once the dynamic assembly among probes was catalyzed by the target DNA, flat ends between two different probes would be created continuously. In this state, a pyrene molecule on one probe was brought into close-enough proximity to another pyrene molecule on the neighboring probe, resulting in the formation of pyrene excimers, which emit at ∼488 nm with 340 nm excitation. Thus, accompanied by dynamic assembly, the switching of pyrene excimers was circularly catalyzed by the target DNA. In addition, in order to provide a further amplified fluorescent signal of the pyrene excimer itself, gammacyclodextrin (γ-CD) was used to modulate the space proximity of two hydrophobic pyrene molecules labeled on a flat end through pyrene/cyclodextrin inclusion interaction, because cyclodextrins are hydrophobic inside and hydrophilic outside.11 Therefore, a fluorescent turn-on enzyme-free isothermal amplification strategy based on target-catalyzed dynamic assembly of branched junction was constructed, and the nucleic acid target might be detected through the fluorescence change of pyrene excimers.
Scheme 1. Schematic Representation for the Proposed Enzyme-Free Nucleic Acid Amplified Detection (T = Target DNA, A = Probe A, B = Probe B, C = Probe C, CD = Cyclodextrin, = Pyrene Molecule)a
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EXPERIMENTAL SECTION Chemicals and Materials. Oligonucleotides involved in this study were synthesized by Takara Bio, Inc. (Dalian, China), and three pyrene-labeled probes were also modified by Takara Bio, Inc.; all DNAs were purified by high-performance liquid chromatography (HPLC). The detailed sequence information on all DNAs is listed in Table 1. The DNA stock solutions of 10 μM were prepared by dissolving oligonucleotides in sterilized hybridization buffer (30 mM Tris-HCl, 300 mM KCl, and 4 mM MgCl2, pH 7.8). Probes were heated to 95 °C for 5 min and then allowed to cool to room temperature for at least 2 h before use. RPMI 1640 cell medium was obtained from Shanghai Sangon Biotechnology Company, Ltd. (China). Fetal bovine serum (FBS) was obtained from Zhejiang Tianhang Biological Technology Co., Ltd. (China). The gamma-cyclodextrin (γ-CD) and salts reagent of analytical reagent grade were commercially obtained from Dingguo Biotechnology Co., Ltd. (China), and were used without further purification or treatment. Corresponding solutions were prepared using deionized water, which was obtained through the Nanopure Infinity ultrapure water system (Barnstead/ Thermolyne Corp.). Electrophoresis Demonstration of Assembly Steps. Different mixtures of target DNA with probes were incubated for 3 h at 10 °C, the concentration of each oligonucleotide was 500 nM. After samples were prepared, 10 μL of each sample was mixed with 2 μL of 6× loading buffer, then 10 μL of mixed solution was added into the gel for electrophoresis. A 15% native polyacrylamide gel was prepared using 1× TBE buffer (89 mM Tris Borate, 2.0 mM EDTA, pH 8.3). The gel was run at 100 V for 2 h in 1× TBE buffer, and stained for 20 min in a 1× SYBR Gold solution (30 mL of H2O, 3 μL of 10000× SYBR Gold), then illuminated with ultraviolet (UV) light and finally photographed by the Tanon-2500R gel imaging system (Shanghai, China). Fluorescence Measurements. All fluorescence measurements were carried out on a F2500 fluorescence spectrophotometer (Hitachi, Japan) equipped with an aqueous thermostat (Amersham) accurate to 0.1 °C. The excitation wavelength was set at 340 nm, Ex and Em slits were both set at 5.0 nm with a
a
Each segment that is labeled with a lowercase letter is six nucleotides in length, and asterisks denote complementary relationships.
Table 1. Oligonucleotides Used in the Study type probe A probe B probe C target DNA mismatched DNA detected DNA inserted DNA random DNA
sequence (from 5′ to 3′) Pyrene-ATTGGA-GCTTGA-GATGTT-AGGGAGTAGTGC-TCCAAT-CACAAC-GCACTA-CTCCCTAACATC-Pyrene Pyrene-GATGTT-AGGGAG-TAGTGC-GTTGTGATTGGA-AACATC-TCAAGC-TCCAAT-CACAACGCACTA-Pyrene Pyrene-TAGTGC-GTTGTG-ATTGGA-GCTTGAGATGTT-GCACTA-CTCCCT-AACATC-TCAAGCTCCAAT-Pyrene GCACTA-CTCCCT-AACATC-TCAAGC GCACTA-CTCCTT-AACATC-TCAAGC GCACTA-CTCC_T-AACATC-TCAAGC GCACTA-CTCCCAT-AACATC-TCAAGC AAAAAG-GAAAGG-GGGGAC-TCACTA-TA
rationally designed with a stem of 18 pairs and an additional 12 nucleotides sticky end at the 5′-end. The complementary relationships between segments of A, B, and C were specified so that the hairpins were kinetically handicapped from crossopening in the absence of the target DNA, and a cascade of assembly steps were catalyzed to form a branched junction as introduction of the target DNA. Each assembly circle was terminated by a disassembly step in which probe C displaced the target DNA from the branched complex, freeing the target DNA to catalyze the next assembly circle. Skillfully, for expedient signal readout, a spatially sensitive fluorescent molecule pyrene was chosen as the signal unit and labeled at both ends of each probe.7a,10 In the absence of self-assembly, all probes were in the closed hairpin formation, the two pyrene molecules labeled on the same probe were spatially separated by the extra length of the 12 nucleotides sticky end. In this state, no excimer fluorescence could be observed because the 4935
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PMT voltage of 700 V. Fluorescence emission spectra were then obtained with a 0.2 cm × 1 cm quartz cuvette containing 200 μL of solution. Assembly Temperature Optimization. The assembly temperature was optimized through the signal-to-background ratio (SBR) parameter. The pyrene excimer fluorescence intensity of the mixture of three probes and the target DNA was recorded after assembly reaction under different temperatures (4, 10, 15, and 20 °C). The concentration of each oligonucleotide was 500 nM, the assembly reaction volume was 200 μL, the assembly reaction time was 3 h, and the recorded fluorescence intensity value was denoted as F. The background fluorescence intensity under different temperature was recorded as the same as the above procedure, except for the absence of the target DNA; the recorded background value was denoted as F0. Then, the SBR was calculated by the formula
SBR =
Figure 1. Effect of temperature on the signal-to-background ratio (SBR) (SBR = F/F0, where F is the fluorescence emission intensity of pyrene excimers at 488 nm in the presence of target DNA, and F0 is the fluorescence emission intensity of pyrene excimers at 488 nm in the absence of target DNA).
F F0
signal; in addition, the thermal stability of the hairpin conformation of probes was weaker at the higher temperature, resulting in more background signal. Thus, 10 °C was chosen as the optimal temperature for the model detection system and was used in further experiments. As described in the schematic graph, the assembly of each branched junction underwent four cross-opening steps, which were confirmed by native gel electrophoresis. As shown in Figure 2, the probes assembled scarcely in the absence of the
Real-Time Monitoring of the Dynamic Assembly. A mixture of 200 μL of three probes and target DNA was added into a quartz cuvette, which was then placed in the fluorescence spectrophotometer and incubated at 10 °C. From the start of the assembly reaction, the resulting excimer fluorescence was recorded every several minutes. The concentration of each oligonucleotide was 500 nM. The temperature was controlled by an aqueous thermostat. The control experiment was the same as the above procedure, except for the absence of the target DNA. Detection Procedure. The detection experiments were performed in 200 μL of a hybridization buffer (30 mM TrisHCl, 300 mM KCl, and 4 mM MgCl2, pH 7.8) consisting of 500 nM probe A, 500 nM probe B, and 500 nM probe C at 10 °C. The target DNA was added into the detection system to catalyze the assembly reaction. After incubation of 3 h, 1 mM γCD was introduced to the resulting solution and incubated for another 5 min. Finally, the resulted solutions were characterized by fluorescence spectrophotometer and the excimer fluorescence was recorded. Preparation of Complex Samples. To test the capability of the proposed strategy for detecting the target DNA from complex biological samples, RPMI 1640 cell medium supplemented with 15% fetal bovine serum, a cell medium extensively used as a model for preparation of complex biological samples,7a,12 was first spiked with the target DNA of different concentrations. Then, the detection procedure according to the proposed strategy was carried out, and the recovery was finally calculated.
Figure 2. Electrophoresis demonstrating the target-catalyzed assembly steps of the formation of branched junctions depicted in Scheme 1.
target DNA (lane 5), while obvious assembly steps were observed as a function of the introduction of the target DNA (lanes 1−4). Interestingly, the band of dissociative probes disappeared after target-catalyzed assembly (lane 4), while a clear band of dissociative probes was still observed after annealing of the mixture of three probes (lane 6), meaning that there were many unassembled probes in this case. This indicated that the target-catalyzed dynamic assembly was more efficient than that observed via the traditional annealing approach. Thus, these results demonstrated that the dynamic assembly was circularly catalyzed by the target DNA, and an enzyme-free amplification strategy might be developed by taking advantage of the isothermal signal-amplification capability of target-catalyzed dynamic assembly. In addition, to confirm the switching of pyrene excimers accompanied by the assembly process, real-time monitoring of
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RESULTS AND DISCUSSION Since temperature can influence the free energy and stability of hybridization, it was first optimized to make the detection system exhibit a better signal-to-background ratio (SBR) (recall that SBR = F/F0, where F is the fluorescence emission intensity of pyrene excimers at 488 nm in the presence of the target DNA, and F0 is the fluorescence emission intensity of pyrene excimers at 488 nm in the absence of the target DNA). As shown in Figure 1, a better SBR is exhibited for the proposed model detection system at 10 °C, while a worse SBR is observed at lower and higher temperature. Those observations were because the hairpin conformation of probes was too stable to cross-open at the lower temperature, resulting in less positive 4936
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the pyrene excimer fluorescence was carried out at the optional temperature (10 °C) (see Figure 3). The fluorescence emission
Figure 3. Real-time monitoring of the changes in the pyrene excimer fluorescence (●) in the presence of the target DNA or (○) with no target DNA at λem = 488 nm with the excitation wavelength of 340 nm. F is the fluorescence emission intensity at the corresponding time point, and F0 is the fluorescence emission intensity at the start of the assembly reaction. Inset shows fluorescence emission images with UV irradiation.
intensity of pyrene excimers increased gradually along with the assembly time in the presence of the target DNA, and reached a plateau at ∼3 h, which was used as the optimal assembly time in future experiments (solid circle in Figure 3). However, negligible fluorescence increment was observed in the absence of the target DNA (hollow circle in Figure 3). Also, when we viewed the resulting solutions under UV light, a clear green color was observed by the naked eye when the target DNA was introduced into the detection system (right-hand side of inset in Figure 3), while little background signal was exhibited in the absence of the target DNA (left-hand side of inset in Figure 3). These results, accompanied by the target-catalyzed dynamic assembly of branched junctions, indicated that the switching of the pyrene excimers successfully occurred. Thus, a fluorescent turn-on enzyme-free isothermal amplification strategycombining the target-catalyzed dynamic assembly of branched junction with the spatially sensitive fluorescent signal of pyrene excimerswas constructed, and the nucleic acid target might be detected through the fluorescence change of pyrene excimers. To achieve a further amplified fluorescent signal of the pyrene excimer itself, a space modulater (γ-CD) of the hydrophobic pyrene excimer of aromatic hydrocarbons was introduced at the end of the assembly reaction. The effect of γCD on signal changes was investigated and is shown in Figure 4A. Although the introduction of γ-CD caused a slight increase of background fluorescence (blue curve), compared to that in the absence of γ-CD (pink curve), fortunately, a much larger SBR was achieved by the introduction of γ-CD. In the absence of γ-CD, an SBR value of only ∼300% was observed for the 500 nM target DNA. In contrast, a SBR of ∼500% was observed for the 500 nM target DNA by introducing γ-CD modulation. In accordance with the reported results,11 our data demonstrated that pyrene excimers could become embedded into a γ-CD cavity (internal diameter of 0.85 nm)13 by hydrophobic interaction, resulting in a further amplified fluorescent signal of the pyrene excimer itself. In order to achieve a better sensing performance, the concentration of γ-CD was also optimized
Figure 4. (A) Pyrene excimer fluorescence spectra of the mixture of the three probes under different conditions. All oligonucleotides had a concentration of 500 nM. γ-CD was present at a concentration of 1 mM. (B) Effect of the γ-CD concentration on the SBR, where F is the fluorescence emission intensity of pyrene excimers at 488 nm in the presence of the target DNA, and F0 is the fluorescence emission intensity of pyrene excimers at 488 nm in the absence of target DNA. The concentration of oligonucleotides was the same as that mentioned previously.
(see Figure 4B), and 1 mM γ-CD was chosen as the optimal concentration and used in the following experiments. Under optimal conditions, to evaluate the response range and detectable minimum concentration of the target DNA by the pyrene excimer fluorescence, the excimer fluorescent signal changes, in response to different concentrations of the target DNA, were measured and are shown in Figure 5A. There was a gradual increase in the fluorescence intensity of pyrene excimers with the increase in the concentration of the target DNA. It should be noted that the minimum concentration of 10 pM target DNA could induce a distinguishable response from the background signal, so 10 pM was defined as the detectable minimum target concentration of the detection system. The relationship between the intensity change of the excimer fluorescence and the concentration of the target DNA was demonstrated in Figure 5B; the standard deviation was obtained from three repeated experiments. A good linear relationship (R2 = 0.994) was obtained in the range from 100 pM to 400 nM for the target DNA (see the inset in Figure 5B). This detection capability was comparable to or even better than some reported HCR-based and enzyme-based methods for DNA amplified detection.7,8g,14 Simultaneously, the selectivity of the proposed DNA detection strategy was carefully evaluated. Several control DNAs, including mismatched DNA, deleted DNA, inserted DNA, and random DNA, were detected by the method instead of the target DNA. As shown in Figure 6, the fluorescence intensity change of pyrene excimers was 4937
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observed in the presence of the target DNA than that in the presence of any mutant DNA, which indicated that our proposed strategy also had the potential to distinguish single nucleotide polymorphism (SNP).15 Therefore, based on these above results, the constructed enzyme-free isothermal amplification strategy could be used to detect the target nucleic acid with good sensitivity and specificity. Detection of the target DNA from complex samples was carried out to test the practical application capability of the proposed strategy. In this work, RPMI 1640 cell medium containing 15% fetal bovine serum was selected as the complex fluid, and the target DNA-spiked complex fluids were detected by the procedure according to the proposed strategy. The recoveries were calculated and demonstrated in Table 2. For Table 2. Detection of the Target DNA from RPMI 1640 Cell Medium added (nM)
detected (nM)
recovery (%)
standard deviation, SD (%)a
10 50 100 250
11.24 49.22 102.84 245.53
112.36 98.45 102.84 98.21
5.10 12.99 7.25 11.57
a
n = 3.
different concentrations of the target DNA in the complex fluids, satisfactory recoveries and acceptable standard deviations were obtained, which suggested that the strategy held the potential for reliable and practical detection of the target DNA from complex fluids.
Figure 5. (A) Fluorescence spectra of the detection system in the presence of target DNA with different concentrations (from bottom to top: 0 pM, 10 pM, 100 pM, 1 nM, 10 nM, 50 nM, 100 nM, 250 nM, 400 nM, 500 nM, 1000 nM). The inset shows the fluorescence responses to the target DNA at low concentration. (B) The relationship between the intensity change of the pyrene excimer fluorescence (at 488 nm) and the target DNA concentration. The inset shows the linear response from 100 pM to 400 nM.
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CONCLUSIONS In summary, a new path for enzyme-free nucleic acid amplified detection has been opened up in this work by combining the isothermal signal-amplification capability of target-catalyzed dynamic assembly with the spatially sensitive fluorescent signal of pyrene excimers. Three metastable pyrene-labeled hairpin DNA probes were designed as assembly components, which were kinetically handicapped from cross-opening in the absence of the target DNA. The introduction of the target DNA could dynamically catalyze the assembly of branched junctions with the switching of pyrene excimers, resulting in a turn-on excimer fluorescence. It was a simple mix-and-detect amplification method, which relied only on hybridization and strandexchange reactions, without requiring expensive and perishable protein enzymes, complicated instrumentations, and complex thermal-cycling procedures. This strategy achieved a good detection capability, which was comparable to or even better than some reported enzyme-dependent amplification methods, and the potential for target detection from complex fluids was also verified. Furthermore, as a novel transformation of dynamic DNA self-assembly technology into enzyme-free signal-amplification analytical application, we infer that the proposed strategy will be useful and helpful for a wide range of fields, such as in vivo imaging, nucleic acid-related detection of diseases and environmental monitoring, and even aptamerbased detection of non-nucleic acid targets.
Figure 6. Responses of intensity changes of pyrene excimer fluorescence to the target DNA, mismatched DNA, deleted DNA, inserted DNA, and random DNA. The concentration used for all detected DNA was 50 nM.
strong and outstanding in the presence of the target DNA, while negligible change was observed in the presence of the random DNA, which indicated that the proposed strategy was capable of detecting the target DNA from random nucleic acid library. Furthermore, at the same detected concentration, although the mismatched DNA, deleted DNA, and inserted DNA could result in a minor change in the pyrene excimer fluorescence, much more fluorescence enhancement was
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AUTHOR INFORMATION
Corresponding Authors
*Tel.: +86-731-88821566. Fax: +86-731-88821566. E-mail:
[email protected] (X. He). 4938
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*Tel.: +86-731-88821566. Fax: +86-731-88821566. E-mail:
[email protected] (K. Wang).
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by the Key Project of Natural Science Foundation of China (Grant Nos. 21175039, 21322509, 21305035, 21190044, and 21221003), Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20110161110016), the project supported by Hunan Provincial Natural Science Foundation and Hunan Provincial Science and Technology Plan of China (No. 2012TT1003), and the project supported by Hunan Provincial Innovation Foundation for Postgraduate.
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