Biosensors and Bioelectronics 59 (2014) 276–281
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation Jing Zhu a, Yongshun Ding a, Xingti Liu a, Lei Wang b,n, Wei Jiang a,nn a Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China b School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 January 2014 Received in revised form 18 March 2014 Accepted 20 March 2014 Available online 1 April 2014
Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for singlebase mutations in biological samples. & 2014 Elsevier B.V. All rights reserved.
Keywords: Single-base mutation Toehold-mediated strand displacement reaction Isothermal DNA amplification Fluorescent detection
1. Introduction DNA single base substitution, insertion, and deletion, also known as single-base mutations, are aberrations widely discovered in genomic DNA of various types of cancers and genetic diseases (Song et al., 2013; Zhu et al., 2009). Often, these singlebase mutations are regarded as valuable molecular markers for cancer diagnostics and particular genetic disorders prognostics owing to their direct connections with transcriptional regulation or biological functions of many proteins (Collins et al., 2003; Kruglyak and Nickerson, 2001; Hirschhorn and Daly, 2005). The development of accurate single-base mutations detection methods become an important issue. Typical assays utilized a key singlen
Corresponding author. Tel.: þ 86 531 88380036. Corresponding author. Tel.: þ 86 531 88362588; fax: þ86 531 88564464. E-mail addresses: [email protected] (L. Wang), [email protected] (W. Jiang). nn
http://dx.doi.org/10.1016/j.bios.2014.03.051 0956-5663/& 2014 Elsevier B.V. All rights reserved.
nucleotide-specific enzymatic reaction, which includes primer extension (Litos et al., 2007) and invasive cleavage (Chen et al., 2005) as well as ligation (Ogasawara and Fujimoto, 2006), to identify the mismatch site within their DNA substrates. These strategies, though exhibit high fidelity in single-base differentiation, still have not prevailed owing to some disadvantageous operational attributes involving sophisticated instrumentation, or multistep washing and separation. Hybridization-based methods are the simplest mechanism for mismatch differentiation (Huang et al., 2013). Although the Watson–Crick paired nucleotides are one of the most specific recognition events, the direct hybridization can not achieve single-base mismatch differentiation. To enhance the discrimination capability, hairpin and other structural elements are employed into the probe molecule to make binding both perfectly matched and single-base mismatched targets with energetic differences (Wang et al., 2009; Xiao et al., 2009). Such energetic differences are often small and can result in strong differences in the hybridization yield. Hence, the
J. Zhu et al. / Biosensors and Bioelectronics 59 (2014) 276–281
mechanism needs to exploit a sophisticated probe design algorithms and introduce hybridization enhancing moieties. Often, rigorous control of the assay conditions is also required for optimal performance (Lin et al., 2011; Zhou et al., 2011). Hence, it remains necessary to develop a highly selective strategy with no need of multistep operations or complex design for single-base mutation detection. Toehold-mediated strand displacement reaction (TSDR), as one of the most powerful strategies to construct and control dynamic DNA nanostructures and nanodevices (Zhang and Seelig, 2011), has been also proven to be successful for highly selective recognition of single-base mutations (Zhang et al., 2010; Wang et al., 2012). This approach overcomes the small differences of free energy between the probe and target binding, which discriminates single-base variations through a kinetic mechanism. In this mechanism, the target sequence hybridizes first with a short single-stranded domain termed “toehold” attached to a prehybridized duplex DNA and a branch migration process occurs in succession. The toehold domain typically includes 5–8 nucleotides, the length and sequence composition of which can be utilized to adjust the kinetic rate by a factor of 106 (Zhang and Winfree, 2009). Hence, a single-base bulge on a toehold could inhibit the progress of branch migration greatly in the direction of the mispair (Genot et al., 2011; Li et al., 2002). Indeed, a hybridized duplex containing a toehold domain as the discrimination probe possesses a desirable selectivity comparable to the traditional molecule beacons (Zhang et al., 2010; Subramanian et al., 2011). Besides the prominent discrimination capability, the sensitivity is also an important factor which determines the analytical performance for the detection of single-base mutation. Recently, some strategies combining TSDR with the isothermal amplification system such as exonuclease III (Exo III) assisted target recycle were reported (Huang et al., 2012; Xu et al., 2012). However, Exo III can also digest some closed hairpin beacon and ssDNA and result in a high background and unsatisfied sensitivity. Additionally, the requirement of fluorescent labeling or base modification increases the experimental cost and the design complexity. To address the above problems, we combine coupled polymerization/nicking enzyme processes that release G-quadruplex sequences as the reporter with TSDR, present a highly sensitive and selective fluorescent detection method for single-base mutation. In this method, the discrimination probe with a toehold at the 30 end was designed to hybridize with the target DNA, and unfolded the stem-loop structure through the strand migration process. Subsequently, the target DNA as a primer induced two consecutive replication and scission as well as strand displacement reactions in the presence of polymerase, nicking enzyme and reporter probe. Such a process released a great amount of G-quadruplex sequences, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. The fluorescence insensitivity was recorded to detection single-base mutations. As proof of concept, we selected the target sequence from the human KRAS gene as a model which was a diagnostic biomarker for a number of cancers including pancreatic, colorectal, and lung cancer (Sidransky, 2002; Ho et al., 2004; Amado et al., 2008). Furthermore, it could be performed in homogeneous format without washing and separation steps as well as fluorescent labeling, making it more simple and cost effective.
2. Experimental section 2.1. Chemicals and materials Oligonucleotides used in this work were synthesized and purified by Invitrogen Biotechnology Co. Ltd. (Shanghai, China),
277
and their sequences were given in Table S1. The DNA stock solution was prepared with the buffer containing 10 mM Tris– HCl and 1 mM EDTA (pH 8.0).Vent (exo-) polymerase, Nt.BstNBI nicking enzyme, 10 Thermopol buffer, 10 NEBuffer 3 were purchased from New England Biolabs, Inc. (Beverly, MA, USA). The deoxynucleotide triphosphates (dNTPs) were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). N-methyl mesoporphyrin IX (NMM) was purchased from J&K Scientific Ltd. (Beijing, China). All other reagents were of analytical grade and used as received. The ultrapure water was used to prepare all of the solutions which was obtained from a Millipore Milli-Q water purification system (418.25 MΩ). 2.2. Toehold-mediated strand displacement reaction triggered isothermal DNA amplification The toehold-mediated strand displacement reaction was performed at 37 1C for 1.5 h in 20 μL reaction buffer (20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% TritonX100) containing 100 nM discrimination probe, 60 nM reporter probe and target DNA at different concentrations. After the branch migration reaction, dNTPs (10 mM), Nt.BstNBI nicking enzyme (0.4 U μL 1), Vent (exo-) polymerase (0.16 U μL 1), 1 ThermoPol buffer (20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100), and 0.5 NEBuffer 3 (25 mM Tris–HCl, pH 7.9, 50 mM NaCl, 5 mM MgCl2, 0.5 mM DTT) were added. Then the isothermal DNA amplification reaction in a volume of 40 μL was carried out at 60 1C for 1.5 h. 2.3. Fluorescence measurements of the DNA analyte After the isothermal DNA amplification reaction, 2 μL NMM (5 μM) and 8 μL KCl (1 M) was added to the resulting product and incubated at 37 1C for 30 min. The fluorescence intensity of the mixture solution was measured on a Hitachi F-7000 spectrofluorophotometer (Hitachi, Japan). The excitation wavelength of NMM was centered at 399 nm (Hu et al., 2011).The fluorescence intensity in the measurement culminated at 609 nm. Thus, the instrument was operated under the following parameters: λex ¼ 399 nm (bandpass 10 nm), λem ¼609 nm (bandpass 10 nm), PMT detector voltage¼550 V. 2.4. Preparation of HeLa cells lysate The HeLa cell samples were pelleted by centrifugation (5 min, 3000 rpm, 4 1C) and resuspended in 20 mL of 10 mM Tris–HCl buffer (pH 7.0) for lysis on ice using a sonicator (four pulses at 200 W for 30 s with a tapered microtip). The mixture solution was then centrifuged at 12,000 rpm for 30 min at 4 1C to remove insoluble material. The resulting supernatant was collected and filtered through a 0.45 mm filter membranes, yielding crude lysate. 3. Results and discussion 3.1. Rationale of toehold-mediated strand displacement reaction triggered isothermal DNA amplification Our strategy for single-base mutation detection comprises a TSDR followed with an autonomous replication-scission-displacement reaction, as illustrated in Scheme 1. Two probes are designed with the stem-loop structures which are denoted as discrimination probe (DP) and reporter probe (RP), respectively. DP has a 6-nucleotide single-strand overhanging domain (known as the toehold) and the linked stem strand to hybridize with target
278
J. Zhu et al. / Biosensors and Bioelectronics 59 (2014) 276–281
Scheme 1. Illustration of single-base mutation detection based on TSDR triggered isothermal DNA amplification-based fluorescent method.
DNA, and with a half recognition sequence of (50 -GACTC-30 ) for Nt. BstNBI nicking enzyme in the loop. RP is designed to have a complementary sequence of G-quadruplex DNA at the stem and 50 overhang domain with the identical recognition sequence of (50 GACTC-30 ) for Nt.BstNBI nicking enzyme in the loop. In a typical procedure as path B, the TSDR is initiated by the hybridization between perfect matched DNA (PM) and the toehold domain, and ended with the unfolding of the DP. Subsequently, the PM behaves as a primer to trigger an extension reaction in the presence of vent (exo-) DNA polymerase and dNTPs. The replication of the DP finally yields the duplex strand with a full recognition site for nicking enzyme. Hence, a cycle of nicking enzyme cleavage, polymerase extension, and subsequent replicated strand release is created, indicating that the TSDR proceeds successfully as expected. Then the released strands are annealed with the 30 overhang domain of RP, launching another replication-scissiondisplacement reaction to produce abundant G-quadruplex sequences. In contrast, as shown in path A, the strand migration process is trapped kinetically because the single-base mismatch at the 50 end of the toehold greatly restrains the hybridization between the DP DNA and single-base mutant sequences (SM). Thus the SM can barely initiate the strand migration and then trigger the autonomous replication-scission-displacement reaction. Hence, there are few G-quadruplex sequences released. We utilize the NMM as the signal reporter which is a commercially available unsymmetrical anionic porphyrin characterized by a prominent structural selectivity for G-quadruplex DNA. It exhibits weak fluorescence in free form, but significantly enhanced fluorescence signal via binding to G-quadruplex DNA (Zhao et al., 2012). Through this character, we obtained significant difference of fluorescence response between the perfect matched target and the single-base mutant targets. So the single base mismatch in the target DNA was discriminated successfully. 3.2. Verification of the discrimination capability by fluorescence spectral characteristics To verify the feasibility of the present strategy, we investigated the fluorescence emission spectra under different conditions. As shown in Fig. 1, compared with the weak fluorescence intensity of NMM (curve a), a little increase of the fluorescence response was observed when the target sequence was absent (curve c), which was defined as background signal. The result might be ascribed to nonspecific background amplification which involved RP but unprimed DNA synthesis as well as the formation of RP dimers
Fig. 1. Fluorescence responses under different conditions. (a) NMM, (b) control experiment using DP DNA without RP DNA, (c) negative experiment in the absence of target DNA, (d) control experiment using RP DNA without DP DNA, (e) response in the presence of DP DNA and RP DNA as well as SM target (10 nM), and (f) response in the presence of DP DNA and RP DNA as well as PM target (10 nM).
(Tan et al., 2008). The presence of PM DNA (10 nM) resulted in a significant fluorescence enhancement (curve f). Such an enhanced fluorescence response signified the formation of an abundant G-quadruplex which greatly enhanced fluorescence emission of NMM, evidencing successful strand migration process initiated by PM DNA followed with the generation of G-quadruplex via subsequent isothermal DNA amplification. In contrast, in the presence of SM DNA, a much weak fluorescence change (curve e) compared with background signal was obtained, which indicated the presence of a very small amount of G-quadruplex in the system since SM failed to open the DP with an unsuccessful isothermal DNA amplification. The above results suggested the high selectivity of this strategy for detecting sing-base mismatch in the target DNA. In a further control experiment, we took the DP away from the system, a fluorescent response as low as the background signal was obtained (curve d). This confirmed that the second-step DNA amplification was primed by the product generated in the firststep DNA amplification. Additional control experiment was further performed to evacuate the RP from the system. In this case, we also observed a weak fluorescence response (curve b) comparable
J. Zhu et al. / Biosensors and Bioelectronics 59 (2014) 276–281
to NMM itself, indicating that G-quadruplex was generated in the second-step DNA amplification reaction.
3.3. Optimization of reaction conditions The reaction conditions were investigated using F/F0 as standard in order to obtain the optimal analytical parameters, where F and F0 were the enhanced fluorescence intensities of the G-quadruplex-NMM complex in the presence and absence of PM DNA, respectively. The kinetic rate of the TSDR is mainly based on the length of toehold. It was reported that the toehold length of 6 nt was the smallest for initiating strand migration at the maximum rate in solution (Zhang and Winfree, 2009). However, the influence of toehold length on the kinetics of isothermal DNA amplification assisted strand migration has not been reported yet. Through altering the length of single-strand overhang domain of the DP, just like DP1 to DP5 made the effective toehold length varying from 8 nt to 4 nt, respectively (Table S1). Fig. 2 shows the dependence of the reaction rate on the length difference of the DP. DP1, DP2, and DP3 had an equilibrium time of about 90 min, much shorter than DP4 and DP5. Furthermore, the result indicated that
Fig. 2. The variation tendency of fluorescence responses with different lengths of toehold. The error bars showed the standard deviation of three replicate determinations.
279
DP1, DP2, and DP3 had a similar kinetic rate for the isothermal DNA amplification assisted strand migration and a toehold length of 6 nt generated the maximum kinetic rate with the minimum bases. Hence, DP3 was selected in the following experiments. To achieve the best sensing performance, the concentrations of DP3 DNA, RP DNA, DNA polymerase, nicking enzyme, and NMM were optimized. Fig. S1–S5 depict typical fluorescence response in correlation to these assay conditions. The concentration of DP3 DNA has a crucial effect on the efficiency of the TSDR (Li et al., 2011). Thus, in order to obtain a high efficiency of strand migration between the target and the DP3 DNA, we varied the concentration of DP3 DNA from 10 nM to 120 nM. As shown in Fig. S1, the fluorescence response gradually increased and became almost leveled off at the concentration of 100 nM. Thus, this concentration of DP3 DNA was used throughout subsequent experiments. To ensure the high sensitivity of the assay, the concentration of RP DNA should be optimized carefully. On one hand, low concentration of RP DNA might limit the amplification efficiency because a large number of the product sequences generated in the firststep DNA amplification would hybridize with the RP DNA, then prime the second-step DNA amplification to gain the substantial sensitivity. On the other hand, a high concentration of RP DNA might lead to high background due to the fact of the nonspecific amplification, which might be ascribing to the formation of RP DNA dimmers (Zhang and Zhang, 2012). Thus the concentration of the RP DNA was investigated from 10 nM to 100 nM. Fig. S2 depicts the dependence of fluorescence response on RP DNA concentrations. It was observed that the values of F/F0 achieved a peak when the concentration of RP DNA was 60 nM. As a result, this concentration was selected in the following experiments. The amounts of the vent (exo-) polymerase and nicking enzyme were investigated because the cooperation of them had crucial effect on the efficiency of isothermal DNA amplification (Wang et al., 2011). It could be seen from Fig. S3 that, the maximum fluorescence signal was obtained with a polymerase concentration of 0.16 U μL 1. Similarly, a peak-shape dependence of fluorescence response on nicking enzyme concentrations was also observed (Fig. S4). The maximum value was obtained at the point of 0.4 U μL 1. Thus, the concentrations of the vent (exo-) polymerase and nicking enzyme in subsequent experiments were 0.16 U μL 1 and 0.4 U μL 1, respectively. The influence of NMM concentration on F/F0 was also studied. Experiments indicated that the concentration of the NMM had effect on both the fluorescence intensities of the G-quadruplex-NMM
Fig. 3. (A) Effect of different target DNA concentrations on fluorescence emission spectra;(a) background, (b) 2 pM, (c) 10 pM, (d) 50 pM, (e) 100 pM, (f) 500 pM, (g) 1 nM, (h) 3 nM, (i) 5 nM, (j) 10 nM. (B) Linear relationship between the fluorescence intensity and the concentration of PM DNA on logarithmic scales. Each data point represents an average of 3 measurements (each error bar indicates the standard deviation).
280
J. Zhu et al. / Biosensors and Bioelectronics 59 (2014) 276–281
3.6. Precision and reproducibility As important parameters to assess an assay in practical application, the precision and reproducibility of the proposed method were investigated. It was observed that the strategy exhibited excellent reproducibility due to its homogeneous assay format with simple operations (Wang et al., 2011). Analyzed from the experimental results, the relative standard deviation (RSD) obtained from the same batch were 2.9%, 3.8%, and 3.6% at 5 nM, 0.5 nM and 50 pM target DNA, respectively. And the RSD of three different batches were 3.4%, 5.7% and 4.5% at the above-mentioned concentrations by measuring the same samples in 3 days at the identical experimental conditions. These results indicated that the proposed method possessed acceptable precision and reproducibility. 3.7. Detection of target DNA in spiked sample Fig. 4. Fluorescence response of the method to perfect matched DNA (10 nM), single-base mutant sequences (10 nM) and background. The error bars are standard deviations of three repetitive measurements.
complex in the presence and absence of PM DNA. With the concentration increasing, the fluorescence intensity with PM in the system increased gradually and leveled off at 5 μM. However, the fluorescence intensity without PM in the system also increased relaxedly. It was observed that the values of F/F0 elevated firstly and reached a maximum when the concentration of 5 μM, then F/F0 decreased gradually (Fig. S5). Taking this into account, the optimized concentration of NMM was 5 μM. 3.4. Sensitivity Under the optimal conditions, different concentrations of PM DNA were examined to further characterize the sensitivity and detection range of this sensing strategy. The different fluorescent responses to PM DNA of various concentrations were shown in Fig. 3A. The fluorescence intensities increased remarkably when the concentration of PM DNA was enhanced from 0 to 10 nM. As shown in Fig. 3B, a dynamic increase of the fluorescence intensity was observed along with the target concentrations in a range of 2 pM–10 nM (R¼ 0.9982) on logarithmic scales with a detection limit of 1.8 pM calculated by the triple signal-to-noise method, which has improved more than 5-fold of magnitude as compared with that Exo III assisted target cycle combining with TSDR (Xu et al., 2012). The improved sensitivity might be attributed to the low background response and outstanding amplification capability of polymerization/nicking enzyme assisted isothermal DNA amplification.
To evaluate the applicability of this assay to detect single-base mutation for real samples, the 10% HeLa cells lysate were spiked with the concentrations of 5 nM and 0.5 nM of PM DNA. The obtained recoveries of the two samples were 90% and 87% with RSD of 7.6% and 5.7%, respectively. Meanwhile, no obvious fluorescence response was observed with the unspiked lysate samples. These results were comparable to the reported methods (Wang et al., 2012, 2013), indicating that our strategy for single-base mutation detection was reliable and had the potential for biological sample application.
4. Conclusions In summary, we designed a novel label-free single-base mutation detection method which was based on the TSDR triggered isothermal DNA amplification with several advantages. First, TSDR was applied to furnish this method with a desirable specificity in identifying target KRAS gene fragment against single-base mutant sequences. Second, the use of isothermal DNA amplification enhanced the sensitivity by creating a great abundant of G-quadruplex signal-reporter sequences. With such dramatic signal amplification capability of the isothermal DNA amplification, we obtained a detection limit of 1.8 pM for target DNA. Third, its label-free, homogeneous detection format could be greatly robust, cost-efficient, readily automated, which supported its considerable potential in clinical applications. In view of its simplicity, selectivity, and sensitivity, we expect that this developed strategy here can be a powerful molecular tool for early diagnosis of cancers and associated studies.
Acknowledgments 3.5. Specificity A key indicator for single-base mutation detection is the selectivity. In order to quantitatively characterize the specificity of this system, we chose the discriminant ratio, the net signal gain obtained with PM DNA to that obtained with SM DNA under the same conditions, as the measure. The single base changes of PM DNA from original G to mutant A, C, and T were named as A-SM, C-SM, and T-SM. As shown in Fig. 4, the present strategy exhibited perfect selectivity to PM DNA, with remarkable discriminant ratio values to A-SM, T-SM, and C-SM that are 7.5, 10.5, and 12.4, respectively. These results signified that the developed strategy exhibited sufficient capability of distinguishing the KRAS gene fragment from its single-base mutant sequences by introducing the TSDR.
This project was supported by the National Natural Science Foundation of China (Grant nos. 21175081, 21175082, and 21375078).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.03.051. References Amado, R.G., Wolf, M., Peeters, M., Van Cutsem, E., Siena, S., Freeman, D.J., Juan, T., Sikorski, R., Suggs, S., Radinsky, R., Patterson, S.D., Chang, D.D., 2008. J. Clin. Oncol. 26, 1626–1634. Chen, Y., Shortreed, M.R., Olivier, M., Smith, L.M., 2005. Anal. Chem. 77, 2400–2405.
J. Zhu et al. / Biosensors and Bioelectronics 59 (2014) 276–281
Collins, F.S., Green, E.D., Guttmacher, A.E., Guyer, M.S., 2003. Nature 422, 835–847. Genot, A.J., Zhang, D.Y., Bath, J., Turberfield, A.J., 2011. J. Am. Chem. Soc. 133, 2177–2182. Hirschhorn, J.N., Daly, M.J., 2005. Nat. Rev. Genet. 6, 95–108. Ho, C.L., Kurman, R.J., Dehari, R., Wang, T.L., Shih, Ie.M., 2004. Cancer Res. 64, 6915–6918. Hu, D., Huang, Z.Z., Pu, F., Ren, J.S., Qu, X.G., 2011. Chem. Eur. J. 17, 1635–1641. Huang, J.H., Su, X.F., Li, Z.G., 2012. Anal. Chem. 84, 5939–5943. Huang, Y., Zhu, J., Li, G.Y., Chen, Z.C., Jiang, J.H., Shen, G.L., Yu, R.Q., 2013. Biosens. Bioelectron. 42, 526–531. Kruglyak, L., Nickerson, D.A., 2001. Nat. Genet. 27, 234–236. Li, B.L., Ellington, A.D., Chen, X., 2011. Nucleic Acids Res. 39, e110. Li, Q.G., Luan, G.Y., Guo, Q.P., Liang, J.X., 2002. Nucleic Acids Res. 30, e5. Lin, Z.Y., Yang, W.Q., Zhang, G.Y., Liu, Q.D., Qiu, B., Cai, Z.W., Chen, G.N., 2011. Chem. Commun. 47, 9069–9071. Litos, I.K., Loannou, P.C., Christopoulos, T.K., Traeger-Synodinos, J., Kanavakis, E., 2007. Anal. Chem. 79, 395–402. Ogasawara, S., Fujimoto, K., 2006. Angew. Chem. Int. Ed. 45, 4512–4515. Sidransky, D., 2002. Nat. Rev. Cancer 2, 210–219. Song, Y., Zhang, Y., Wang, T.H., 2013. Small 9, 1096–1105. Subramanian, H.K., Chakraborty, B., Sha, R., Seeman, N.C., 2011. Nano Lett. 11, 910–913. Tan, E., Erwin, B., Dames, S., Ferguson, T., Buechel, M., Irvine, B., Voelkerding, K., Niemz, A., 2008. Biochemistry 47, 9987–9999.
281
Wang, D., Tang, W., Wu, X., Chen, G., Chen, Q., Li, N., Liu, F., 2012. Anal. Chem. 84, 7008–7014. Wang, D., Chen, G., Wang, H., Tang, W., Pan, W., Li, N., Liu, F., 2013. Biosens. Bioelectron. 48, 276–280. Wang, H.Q., Liu, W.Y., Wu, Z., Tang, L.J., Xu, X.M., Yu, R.Q., Jiang, J.H., 2011. Anal. Chem. 83, 1883–1889. Wang, K.M., Tang, Z.W., Yang, C.J., Kim, Y., Fang, X.H., Li, W., Wu, Y.R., Medley, C.D., Cao, Z.H., Li, J., Colon, P., Lin, H., Tan, W.H., 2009. Angew. Chem. Int. Ed. 48, 856–870. Xiao, Y., Lou, X.H., Uzama, T., Plakos, K.J.I., Plaxco, K.W., Soh, H.T., 2009. J. Am. Chem. Soc. 131, 15311–15316. Xu, Q.F., Cao, A.P., Zhang, L.F., Zhang, C.Y., 2012. Anal. Chem. 84, 10845–10851. Zhang, D.Y., Winfree, E., 2009. J. Am. Chem. Soc. 131, 17303–17314. Zhang, D.Y., Seelig, G., 2011. Nat. Chem. 3, 103–113. Zhang, Z., Zeng, D.D., Ma, H.W., Feng, G.Y., Hu, J., He, L., Li, C., Fan, C.H., 2010. Small 6, 1854–1858. Zhang, Z.Z., Zhang, C.Y., 2012. Anal. Chem. 84, 1623–1629. Zhao, J.J., Zhang, L.L., Jiang, J.H., Shen, G.L., Yu, R.Q., 2012. Chem. Commun. 48, 4468–4470. Zhou, H., Liu, J., Xu, J.J., Chen, H.Y., 2011. Anal. Chem. 83, 8320–8328. Zhu, D., Xing, D., Tang, Y., Zhang, L., 2009. Biosens. Bioelectron. 24, 3306–3310.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation Jing Zhu a, Yongshun Ding a, Xingti Liu a, Lei Wang b,n, Wei Jiang a,nn a Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China b School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 2 January 2014 Received in revised form 18 March 2014 Accepted 20 March 2014 Available online 1 April 2014
Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for singlebase mutations in biological samples. & 2014 Elsevier B.V. All rights reserved.
Keywords: Single-base mutation Toehold-mediated strand displacement reaction Isothermal DNA amplification Fluorescent detection
1. Introduction DNA single base substitution, insertion, and deletion, also known as single-base mutations, are aberrations widely discovered in genomic DNA of various types of cancers and genetic diseases (Song et al., 2013; Zhu et al., 2009). Often, these singlebase mutations are regarded as valuable molecular markers for cancer diagnostics and particular genetic disorders prognostics owing to their direct connections with transcriptional regulation or biological functions of many proteins (Collins et al., 2003; Kruglyak and Nickerson, 2001; Hirschhorn and Daly, 2005). The development of accurate single-base mutations detection methods become an important issue. Typical assays utilized a key singlen
Corresponding author. Tel.: þ 86 531 88380036. Corresponding author. Tel.: þ 86 531 88362588; fax: þ86 531 88564464. E-mail addresses: [email protected] (L. Wang), [email protected] (W. Jiang). nn
http://dx.doi.org/10.1016/j.bios.2014.03.051 0956-5663/& 2014 Elsevier B.V. All rights reserved.
nucleotide-specific enzymatic reaction, which includes primer extension (Litos et al., 2007) and invasive cleavage (Chen et al., 2005) as well as ligation (Ogasawara and Fujimoto, 2006), to identify the mismatch site within their DNA substrates. These strategies, though exhibit high fidelity in single-base differentiation, still have not prevailed owing to some disadvantageous operational attributes involving sophisticated instrumentation, or multistep washing and separation. Hybridization-based methods are the simplest mechanism for mismatch differentiation (Huang et al., 2013). Although the Watson–Crick paired nucleotides are one of the most specific recognition events, the direct hybridization can not achieve single-base mismatch differentiation. To enhance the discrimination capability, hairpin and other structural elements are employed into the probe molecule to make binding both perfectly matched and single-base mismatched targets with energetic differences (Wang et al., 2009; Xiao et al., 2009). Such energetic differences are often small and can result in strong differences in the hybridization yield. Hence, the
J. Zhu et al. / Biosensors and Bioelectronics 59 (2014) 276–281
mechanism needs to exploit a sophisticated probe design algorithms and introduce hybridization enhancing moieties. Often, rigorous control of the assay conditions is also required for optimal performance (Lin et al., 2011; Zhou et al., 2011). Hence, it remains necessary to develop a highly selective strategy with no need of multistep operations or complex design for single-base mutation detection. Toehold-mediated strand displacement reaction (TSDR), as one of the most powerful strategies to construct and control dynamic DNA nanostructures and nanodevices (Zhang and Seelig, 2011), has been also proven to be successful for highly selective recognition of single-base mutations (Zhang et al., 2010; Wang et al., 2012). This approach overcomes the small differences of free energy between the probe and target binding, which discriminates single-base variations through a kinetic mechanism. In this mechanism, the target sequence hybridizes first with a short single-stranded domain termed “toehold” attached to a prehybridized duplex DNA and a branch migration process occurs in succession. The toehold domain typically includes 5–8 nucleotides, the length and sequence composition of which can be utilized to adjust the kinetic rate by a factor of 106 (Zhang and Winfree, 2009). Hence, a single-base bulge on a toehold could inhibit the progress of branch migration greatly in the direction of the mispair (Genot et al., 2011; Li et al., 2002). Indeed, a hybridized duplex containing a toehold domain as the discrimination probe possesses a desirable selectivity comparable to the traditional molecule beacons (Zhang et al., 2010; Subramanian et al., 2011). Besides the prominent discrimination capability, the sensitivity is also an important factor which determines the analytical performance for the detection of single-base mutation. Recently, some strategies combining TSDR with the isothermal amplification system such as exonuclease III (Exo III) assisted target recycle were reported (Huang et al., 2012; Xu et al., 2012). However, Exo III can also digest some closed hairpin beacon and ssDNA and result in a high background and unsatisfied sensitivity. Additionally, the requirement of fluorescent labeling or base modification increases the experimental cost and the design complexity. To address the above problems, we combine coupled polymerization/nicking enzyme processes that release G-quadruplex sequences as the reporter with TSDR, present a highly sensitive and selective fluorescent detection method for single-base mutation. In this method, the discrimination probe with a toehold at the 30 end was designed to hybridize with the target DNA, and unfolded the stem-loop structure through the strand migration process. Subsequently, the target DNA as a primer induced two consecutive replication and scission as well as strand displacement reactions in the presence of polymerase, nicking enzyme and reporter probe. Such a process released a great amount of G-quadruplex sequences, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. The fluorescence insensitivity was recorded to detection single-base mutations. As proof of concept, we selected the target sequence from the human KRAS gene as a model which was a diagnostic biomarker for a number of cancers including pancreatic, colorectal, and lung cancer (Sidransky, 2002; Ho et al., 2004; Amado et al., 2008). Furthermore, it could be performed in homogeneous format without washing and separation steps as well as fluorescent labeling, making it more simple and cost effective.
2. Experimental section 2.1. Chemicals and materials Oligonucleotides used in this work were synthesized and purified by Invitrogen Biotechnology Co. Ltd. (Shanghai, China),
277
and their sequences were given in Table S1. The DNA stock solution was prepared with the buffer containing 10 mM Tris– HCl and 1 mM EDTA (pH 8.0).Vent (exo-) polymerase, Nt.BstNBI nicking enzyme, 10 Thermopol buffer, 10 NEBuffer 3 were purchased from New England Biolabs, Inc. (Beverly, MA, USA). The deoxynucleotide triphosphates (dNTPs) were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). N-methyl mesoporphyrin IX (NMM) was purchased from J&K Scientific Ltd. (Beijing, China). All other reagents were of analytical grade and used as received. The ultrapure water was used to prepare all of the solutions which was obtained from a Millipore Milli-Q water purification system (418.25 MΩ). 2.2. Toehold-mediated strand displacement reaction triggered isothermal DNA amplification The toehold-mediated strand displacement reaction was performed at 37 1C for 1.5 h in 20 μL reaction buffer (20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% TritonX100) containing 100 nM discrimination probe, 60 nM reporter probe and target DNA at different concentrations. After the branch migration reaction, dNTPs (10 mM), Nt.BstNBI nicking enzyme (0.4 U μL 1), Vent (exo-) polymerase (0.16 U μL 1), 1 ThermoPol buffer (20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100), and 0.5 NEBuffer 3 (25 mM Tris–HCl, pH 7.9, 50 mM NaCl, 5 mM MgCl2, 0.5 mM DTT) were added. Then the isothermal DNA amplification reaction in a volume of 40 μL was carried out at 60 1C for 1.5 h. 2.3. Fluorescence measurements of the DNA analyte After the isothermal DNA amplification reaction, 2 μL NMM (5 μM) and 8 μL KCl (1 M) was added to the resulting product and incubated at 37 1C for 30 min. The fluorescence intensity of the mixture solution was measured on a Hitachi F-7000 spectrofluorophotometer (Hitachi, Japan). The excitation wavelength of NMM was centered at 399 nm (Hu et al., 2011).The fluorescence intensity in the measurement culminated at 609 nm. Thus, the instrument was operated under the following parameters: λex ¼ 399 nm (bandpass 10 nm), λem ¼609 nm (bandpass 10 nm), PMT detector voltage¼550 V. 2.4. Preparation of HeLa cells lysate The HeLa cell samples were pelleted by centrifugation (5 min, 3000 rpm, 4 1C) and resuspended in 20 mL of 10 mM Tris–HCl buffer (pH 7.0) for lysis on ice using a sonicator (four pulses at 200 W for 30 s with a tapered microtip). The mixture solution was then centrifuged at 12,000 rpm for 30 min at 4 1C to remove insoluble material. The resulting supernatant was collected and filtered through a 0.45 mm filter membranes, yielding crude lysate. 3. Results and discussion 3.1. Rationale of toehold-mediated strand displacement reaction triggered isothermal DNA amplification Our strategy for single-base mutation detection comprises a TSDR followed with an autonomous replication-scission-displacement reaction, as illustrated in Scheme 1. Two probes are designed with the stem-loop structures which are denoted as discrimination probe (DP) and reporter probe (RP), respectively. DP has a 6-nucleotide single-strand overhanging domain (known as the toehold) and the linked stem strand to hybridize with target
278
J. Zhu et al. / Biosensors and Bioelectronics 59 (2014) 276–281
Scheme 1. Illustration of single-base mutation detection based on TSDR triggered isothermal DNA amplification-based fluorescent method.
DNA, and with a half recognition sequence of (50 -GACTC-30 ) for Nt. BstNBI nicking enzyme in the loop. RP is designed to have a complementary sequence of G-quadruplex DNA at the stem and 50 overhang domain with the identical recognition sequence of (50 GACTC-30 ) for Nt.BstNBI nicking enzyme in the loop. In a typical procedure as path B, the TSDR is initiated by the hybridization between perfect matched DNA (PM) and the toehold domain, and ended with the unfolding of the DP. Subsequently, the PM behaves as a primer to trigger an extension reaction in the presence of vent (exo-) DNA polymerase and dNTPs. The replication of the DP finally yields the duplex strand with a full recognition site for nicking enzyme. Hence, a cycle of nicking enzyme cleavage, polymerase extension, and subsequent replicated strand release is created, indicating that the TSDR proceeds successfully as expected. Then the released strands are annealed with the 30 overhang domain of RP, launching another replication-scissiondisplacement reaction to produce abundant G-quadruplex sequences. In contrast, as shown in path A, the strand migration process is trapped kinetically because the single-base mismatch at the 50 end of the toehold greatly restrains the hybridization between the DP DNA and single-base mutant sequences (SM). Thus the SM can barely initiate the strand migration and then trigger the autonomous replication-scission-displacement reaction. Hence, there are few G-quadruplex sequences released. We utilize the NMM as the signal reporter which is a commercially available unsymmetrical anionic porphyrin characterized by a prominent structural selectivity for G-quadruplex DNA. It exhibits weak fluorescence in free form, but significantly enhanced fluorescence signal via binding to G-quadruplex DNA (Zhao et al., 2012). Through this character, we obtained significant difference of fluorescence response between the perfect matched target and the single-base mutant targets. So the single base mismatch in the target DNA was discriminated successfully. 3.2. Verification of the discrimination capability by fluorescence spectral characteristics To verify the feasibility of the present strategy, we investigated the fluorescence emission spectra under different conditions. As shown in Fig. 1, compared with the weak fluorescence intensity of NMM (curve a), a little increase of the fluorescence response was observed when the target sequence was absent (curve c), which was defined as background signal. The result might be ascribed to nonspecific background amplification which involved RP but unprimed DNA synthesis as well as the formation of RP dimers
Fig. 1. Fluorescence responses under different conditions. (a) NMM, (b) control experiment using DP DNA without RP DNA, (c) negative experiment in the absence of target DNA, (d) control experiment using RP DNA without DP DNA, (e) response in the presence of DP DNA and RP DNA as well as SM target (10 nM), and (f) response in the presence of DP DNA and RP DNA as well as PM target (10 nM).
(Tan et al., 2008). The presence of PM DNA (10 nM) resulted in a significant fluorescence enhancement (curve f). Such an enhanced fluorescence response signified the formation of an abundant G-quadruplex which greatly enhanced fluorescence emission of NMM, evidencing successful strand migration process initiated by PM DNA followed with the generation of G-quadruplex via subsequent isothermal DNA amplification. In contrast, in the presence of SM DNA, a much weak fluorescence change (curve e) compared with background signal was obtained, which indicated the presence of a very small amount of G-quadruplex in the system since SM failed to open the DP with an unsuccessful isothermal DNA amplification. The above results suggested the high selectivity of this strategy for detecting sing-base mismatch in the target DNA. In a further control experiment, we took the DP away from the system, a fluorescent response as low as the background signal was obtained (curve d). This confirmed that the second-step DNA amplification was primed by the product generated in the firststep DNA amplification. Additional control experiment was further performed to evacuate the RP from the system. In this case, we also observed a weak fluorescence response (curve b) comparable
J. Zhu et al. / Biosensors and Bioelectronics 59 (2014) 276–281
to NMM itself, indicating that G-quadruplex was generated in the second-step DNA amplification reaction.
3.3. Optimization of reaction conditions The reaction conditions were investigated using F/F0 as standard in order to obtain the optimal analytical parameters, where F and F0 were the enhanced fluorescence intensities of the G-quadruplex-NMM complex in the presence and absence of PM DNA, respectively. The kinetic rate of the TSDR is mainly based on the length of toehold. It was reported that the toehold length of 6 nt was the smallest for initiating strand migration at the maximum rate in solution (Zhang and Winfree, 2009). However, the influence of toehold length on the kinetics of isothermal DNA amplification assisted strand migration has not been reported yet. Through altering the length of single-strand overhang domain of the DP, just like DP1 to DP5 made the effective toehold length varying from 8 nt to 4 nt, respectively (Table S1). Fig. 2 shows the dependence of the reaction rate on the length difference of the DP. DP1, DP2, and DP3 had an equilibrium time of about 90 min, much shorter than DP4 and DP5. Furthermore, the result indicated that
Fig. 2. The variation tendency of fluorescence responses with different lengths of toehold. The error bars showed the standard deviation of three replicate determinations.
279
DP1, DP2, and DP3 had a similar kinetic rate for the isothermal DNA amplification assisted strand migration and a toehold length of 6 nt generated the maximum kinetic rate with the minimum bases. Hence, DP3 was selected in the following experiments. To achieve the best sensing performance, the concentrations of DP3 DNA, RP DNA, DNA polymerase, nicking enzyme, and NMM were optimized. Fig. S1–S5 depict typical fluorescence response in correlation to these assay conditions. The concentration of DP3 DNA has a crucial effect on the efficiency of the TSDR (Li et al., 2011). Thus, in order to obtain a high efficiency of strand migration between the target and the DP3 DNA, we varied the concentration of DP3 DNA from 10 nM to 120 nM. As shown in Fig. S1, the fluorescence response gradually increased and became almost leveled off at the concentration of 100 nM. Thus, this concentration of DP3 DNA was used throughout subsequent experiments. To ensure the high sensitivity of the assay, the concentration of RP DNA should be optimized carefully. On one hand, low concentration of RP DNA might limit the amplification efficiency because a large number of the product sequences generated in the firststep DNA amplification would hybridize with the RP DNA, then prime the second-step DNA amplification to gain the substantial sensitivity. On the other hand, a high concentration of RP DNA might lead to high background due to the fact of the nonspecific amplification, which might be ascribing to the formation of RP DNA dimmers (Zhang and Zhang, 2012). Thus the concentration of the RP DNA was investigated from 10 nM to 100 nM. Fig. S2 depicts the dependence of fluorescence response on RP DNA concentrations. It was observed that the values of F/F0 achieved a peak when the concentration of RP DNA was 60 nM. As a result, this concentration was selected in the following experiments. The amounts of the vent (exo-) polymerase and nicking enzyme were investigated because the cooperation of them had crucial effect on the efficiency of isothermal DNA amplification (Wang et al., 2011). It could be seen from Fig. S3 that, the maximum fluorescence signal was obtained with a polymerase concentration of 0.16 U μL 1. Similarly, a peak-shape dependence of fluorescence response on nicking enzyme concentrations was also observed (Fig. S4). The maximum value was obtained at the point of 0.4 U μL 1. Thus, the concentrations of the vent (exo-) polymerase and nicking enzyme in subsequent experiments were 0.16 U μL 1 and 0.4 U μL 1, respectively. The influence of NMM concentration on F/F0 was also studied. Experiments indicated that the concentration of the NMM had effect on both the fluorescence intensities of the G-quadruplex-NMM
Fig. 3. (A) Effect of different target DNA concentrations on fluorescence emission spectra;(a) background, (b) 2 pM, (c) 10 pM, (d) 50 pM, (e) 100 pM, (f) 500 pM, (g) 1 nM, (h) 3 nM, (i) 5 nM, (j) 10 nM. (B) Linear relationship between the fluorescence intensity and the concentration of PM DNA on logarithmic scales. Each data point represents an average of 3 measurements (each error bar indicates the standard deviation).
280
J. Zhu et al. / Biosensors and Bioelectronics 59 (2014) 276–281
3.6. Precision and reproducibility As important parameters to assess an assay in practical application, the precision and reproducibility of the proposed method were investigated. It was observed that the strategy exhibited excellent reproducibility due to its homogeneous assay format with simple operations (Wang et al., 2011). Analyzed from the experimental results, the relative standard deviation (RSD) obtained from the same batch were 2.9%, 3.8%, and 3.6% at 5 nM, 0.5 nM and 50 pM target DNA, respectively. And the RSD of three different batches were 3.4%, 5.7% and 4.5% at the above-mentioned concentrations by measuring the same samples in 3 days at the identical experimental conditions. These results indicated that the proposed method possessed acceptable precision and reproducibility. 3.7. Detection of target DNA in spiked sample Fig. 4. Fluorescence response of the method to perfect matched DNA (10 nM), single-base mutant sequences (10 nM) and background. The error bars are standard deviations of three repetitive measurements.
complex in the presence and absence of PM DNA. With the concentration increasing, the fluorescence intensity with PM in the system increased gradually and leveled off at 5 μM. However, the fluorescence intensity without PM in the system also increased relaxedly. It was observed that the values of F/F0 elevated firstly and reached a maximum when the concentration of 5 μM, then F/F0 decreased gradually (Fig. S5). Taking this into account, the optimized concentration of NMM was 5 μM. 3.4. Sensitivity Under the optimal conditions, different concentrations of PM DNA were examined to further characterize the sensitivity and detection range of this sensing strategy. The different fluorescent responses to PM DNA of various concentrations were shown in Fig. 3A. The fluorescence intensities increased remarkably when the concentration of PM DNA was enhanced from 0 to 10 nM. As shown in Fig. 3B, a dynamic increase of the fluorescence intensity was observed along with the target concentrations in a range of 2 pM–10 nM (R¼ 0.9982) on logarithmic scales with a detection limit of 1.8 pM calculated by the triple signal-to-noise method, which has improved more than 5-fold of magnitude as compared with that Exo III assisted target cycle combining with TSDR (Xu et al., 2012). The improved sensitivity might be attributed to the low background response and outstanding amplification capability of polymerization/nicking enzyme assisted isothermal DNA amplification.
To evaluate the applicability of this assay to detect single-base mutation for real samples, the 10% HeLa cells lysate were spiked with the concentrations of 5 nM and 0.5 nM of PM DNA. The obtained recoveries of the two samples were 90% and 87% with RSD of 7.6% and 5.7%, respectively. Meanwhile, no obvious fluorescence response was observed with the unspiked lysate samples. These results were comparable to the reported methods (Wang et al., 2012, 2013), indicating that our strategy for single-base mutation detection was reliable and had the potential for biological sample application.
4. Conclusions In summary, we designed a novel label-free single-base mutation detection method which was based on the TSDR triggered isothermal DNA amplification with several advantages. First, TSDR was applied to furnish this method with a desirable specificity in identifying target KRAS gene fragment against single-base mutant sequences. Second, the use of isothermal DNA amplification enhanced the sensitivity by creating a great abundant of G-quadruplex signal-reporter sequences. With such dramatic signal amplification capability of the isothermal DNA amplification, we obtained a detection limit of 1.8 pM for target DNA. Third, its label-free, homogeneous detection format could be greatly robust, cost-efficient, readily automated, which supported its considerable potential in clinical applications. In view of its simplicity, selectivity, and sensitivity, we expect that this developed strategy here can be a powerful molecular tool for early diagnosis of cancers and associated studies.
Acknowledgments 3.5. Specificity A key indicator for single-base mutation detection is the selectivity. In order to quantitatively characterize the specificity of this system, we chose the discriminant ratio, the net signal gain obtained with PM DNA to that obtained with SM DNA under the same conditions, as the measure. The single base changes of PM DNA from original G to mutant A, C, and T were named as A-SM, C-SM, and T-SM. As shown in Fig. 4, the present strategy exhibited perfect selectivity to PM DNA, with remarkable discriminant ratio values to A-SM, T-SM, and C-SM that are 7.5, 10.5, and 12.4, respectively. These results signified that the developed strategy exhibited sufficient capability of distinguishing the KRAS gene fragment from its single-base mutant sequences by introducing the TSDR.
This project was supported by the National Natural Science Foundation of China (Grant nos. 21175081, 21175082, and 21375078).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.03.051. References Amado, R.G., Wolf, M., Peeters, M., Van Cutsem, E., Siena, S., Freeman, D.J., Juan, T., Sikorski, R., Suggs, S., Radinsky, R., Patterson, S.D., Chang, D.D., 2008. J. Clin. Oncol. 26, 1626–1634. Chen, Y., Shortreed, M.R., Olivier, M., Smith, L.M., 2005. Anal. Chem. 77, 2400–2405.
J. Zhu et al. / Biosensors and Bioelectronics 59 (2014) 276–281
Collins, F.S., Green, E.D., Guttmacher, A.E., Guyer, M.S., 2003. Nature 422, 835–847. Genot, A.J., Zhang, D.Y., Bath, J., Turberfield, A.J., 2011. J. Am. Chem. Soc. 133, 2177–2182. Hirschhorn, J.N., Daly, M.J., 2005. Nat. Rev. Genet. 6, 95–108. Ho, C.L., Kurman, R.J., Dehari, R., Wang, T.L., Shih, Ie.M., 2004. Cancer Res. 64, 6915–6918. Hu, D., Huang, Z.Z., Pu, F., Ren, J.S., Qu, X.G., 2011. Chem. Eur. J. 17, 1635–1641. Huang, J.H., Su, X.F., Li, Z.G., 2012. Anal. Chem. 84, 5939–5943. Huang, Y., Zhu, J., Li, G.Y., Chen, Z.C., Jiang, J.H., Shen, G.L., Yu, R.Q., 2013. Biosens. Bioelectron. 42, 526–531. Kruglyak, L., Nickerson, D.A., 2001. Nat. Genet. 27, 234–236. Li, B.L., Ellington, A.D., Chen, X., 2011. Nucleic Acids Res. 39, e110. Li, Q.G., Luan, G.Y., Guo, Q.P., Liang, J.X., 2002. Nucleic Acids Res. 30, e5. Lin, Z.Y., Yang, W.Q., Zhang, G.Y., Liu, Q.D., Qiu, B., Cai, Z.W., Chen, G.N., 2011. Chem. Commun. 47, 9069–9071. Litos, I.K., Loannou, P.C., Christopoulos, T.K., Traeger-Synodinos, J., Kanavakis, E., 2007. Anal. Chem. 79, 395–402. Ogasawara, S., Fujimoto, K., 2006. Angew. Chem. Int. Ed. 45, 4512–4515. Sidransky, D., 2002. Nat. Rev. Cancer 2, 210–219. Song, Y., Zhang, Y., Wang, T.H., 2013. Small 9, 1096–1105. Subramanian, H.K., Chakraborty, B., Sha, R., Seeman, N.C., 2011. Nano Lett. 11, 910–913. Tan, E., Erwin, B., Dames, S., Ferguson, T., Buechel, M., Irvine, B., Voelkerding, K., Niemz, A., 2008. Biochemistry 47, 9987–9999.
281
Wang, D., Tang, W., Wu, X., Chen, G., Chen, Q., Li, N., Liu, F., 2012. Anal. Chem. 84, 7008–7014. Wang, D., Chen, G., Wang, H., Tang, W., Pan, W., Li, N., Liu, F., 2013. Biosens. Bioelectron. 48, 276–280. Wang, H.Q., Liu, W.Y., Wu, Z., Tang, L.J., Xu, X.M., Yu, R.Q., Jiang, J.H., 2011. Anal. Chem. 83, 1883–1889. Wang, K.M., Tang, Z.W., Yang, C.J., Kim, Y., Fang, X.H., Li, W., Wu, Y.R., Medley, C.D., Cao, Z.H., Li, J., Colon, P., Lin, H., Tan, W.H., 2009. Angew. Chem. Int. Ed. 48, 856–870. Xiao, Y., Lou, X.H., Uzama, T., Plakos, K.J.I., Plaxco, K.W., Soh, H.T., 2009. J. Am. Chem. Soc. 131, 15311–15316. Xu, Q.F., Cao, A.P., Zhang, L.F., Zhang, C.Y., 2012. Anal. Chem. 84, 10845–10851. Zhang, D.Y., Winfree, E., 2009. J. Am. Chem. Soc. 131, 17303–17314. Zhang, D.Y., Seelig, G., 2011. Nat. Chem. 3, 103–113. Zhang, Z., Zeng, D.D., Ma, H.W., Feng, G.Y., Hu, J., He, L., Li, C., Fan, C.H., 2010. Small 6, 1854–1858. Zhang, Z.Z., Zhang, C.Y., 2012. Anal. Chem. 84, 1623–1629. Zhao, J.J., Zhang, L.L., Jiang, J.H., Shen, G.L., Yu, R.Q., 2012. Chem. Commun. 48, 4468–4470. Zhou, H., Liu, J., Xu, J.J., Chen, H.Y., 2011. Anal. Chem. 83, 8320–8328. Zhu, D., Xing, D., Tang, Y., Zhang, L., 2009. Biosens. Bioelectron. 24, 3306–3310.
