Selective and Homogeneous Fluorescent DNA Detection by Target

Poly{(1,4-phenylene)-2,7-[9,9-bis(6'-N,N,N-trimethylammonium)-hexyl fluorene]dibromide} (CCP-1) was synthesized according to the procedure in the lite...
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Anal. Chem. 2008, 80, 2239-2243

Selective and Homogeneous Fluorescent DNA Detection by Target-Induced Strand Displacement Using Cationic Conjugated Polyelectrolytes Fang He, Fude Feng, Xinrui Duan, Shu Wang,* Yuliang Li,* and Daoben Zhu

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China

A new methodology has been developed for DNA detection that interfaces optical amplification properties of cationic conjugated polyelectrolytes with highly selective targetinduced DNA strand displacement. The probe solution contains a cationic conjugated polyelectrolyte (CCP-1), partly hybridized duplex DNA labeled with a fluorescein at the 5′-terminus, and endonuclease Hae III. Excitation of the CCP-1 leads to efficient energy transfer from CCP-1 to fluorescein. In the presence of a complementary DNA strand to one strand of the probe duplex, a hairpin DNA with the recognition site of endonuclease Hae at the double-stranded stem is released following its cleavage by Hae III to generate short DNA fragment carrying fluorescein. The relatively weak electrostatic interactions between the DNA fragment and CCP-1 lead fluorescein far away from CCP-1 and inefficient energy transfer between them is present. Thus, the DNA can be detected by fluorescence spectra in view of the observed CCP-1 or fluorescein emission changes in aqueous solutions. To avoid utilizing unstable Hae III endonuclease, a new system based on RNA-cleaving DNAzyme was further developed. The protocol offers a convenient approach for homogeneous, selective, and sensitive DNA assay in aqueous solution without using any denaturation steps. Compared with previously reported DNA sensors based on conjugated polyelectrolytes, our new method is highly sequence specific and a single-nucleotide mismatch can be clearly detected in target DNA. Recently the use of conjugated polymers (CPs) as optical platforms in biosensors has attained significant interest.1-9 The signal amplification property of CPs by a collective optical response imparts the sensor high sensitivity and therefore offers a key * Corresponding author. E-mail: [email protected]. (1) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339. (2) Liu, B.; Bazan, G. C. Chem. Mater. 2004, 16, 4467. (3) Ho, H. A.; Be´ra-Abe´rem, M.; Leclerc, M. Chem.sEur. J. 2005, 11, 1718. (4) Bunz, U. H. F. Chem. Rev. 2000, 100, 1605. (5) Chen, L.; McBranch, D. W.; Wang, H. L.; Hegelson, R.; Wudl, F.; Whitten, D. C. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12287. (6) Nilsson, K. P. R.; Ingana¨s, O. Nat. Mater. 2003, 2, 419. (7) Pinto, M. R.; Schanze, K. S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7505. (8) Feng, F.; Tang, Y.; Wang, S.; Li, Y.; Zhu, D. Angew. Chem., Int. Ed. 2007, 46, 7882. (9) Duan, X.; Li, Z.; He, F.; Wang, S. J. Am. Chem. Soc. 2007, 129, 4154. 10.1021/ac702415p CCC: $40.75 Published on Web 02/09/2008

© 2008 American Chemical Society

advantage over sensors based on small molecules.10,11 Because of the importance of DNA detection in clinical diagnosis, gene expression analysis, and other biomedical studies,12,13 the CPs have been utilized to develop new fluorescent platforms for DNA detection.14-16 In these assays, the CPs transduce the hybridization event of single-stranded probe and target DNA to optical signal with high sensitivity. However, the selectivity of these DNA sensors is not satisfactory, and a single-nucleotide mismatch in the target sequence cannot be discriminated as a result of the nonspecific electrostatic interactions between CPs and the probe DNA. To circumvent the limitation, microspheres, melting curves, or PNA probe-assisted DNA detection by using CPs have been developed,17-21 however they either need multiple steps, high temperature, sophisticated process or expensive PNA. Therefore, a new, convenient, selective, and homogeneous method for DNA detection using CPs is still needed. Although DNA hybridization is the most specific recognition event in DNA detection,22 it is difficult to achieve mismatch discrimination. To overcome this disadvantage, a displacement hybridization event has been used to improve the selectivity of the DNA sensors,23,24 where an additional ssDNA competitor is included to form a stable duplex with the probe in the absence of the target DNA and can be displaced in the presence of the target DNA. Here, we use CPs to transduce target-induced DNA strand (10) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev. 2000, 100, 2537. (11) Swager, T. M. Acc. Chem. Res. 1998, 31, 201. (12) Heller, M. J. Annu. Rev. Biomed. Eng. 2002, 4, 129. (13) Schork, N. J.; Fallin, D.; Lanchbury, J. S. Clin. Genet. 2000, 58, 250. (14) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954. (15) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2003, 125, 896. (16) Wang, S.; Gaylord, B. S.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 5446. (17) Kushon, S. A.; Ley, K. D.; Bradford, K.; Jones, R. M.; McBranch, D.; Whitten, D. Langmuir 2002, 18, 7245. (18) Xu, H.; Wu, H.; Huang, F.; Song, S.; Li, W.; Cao, Y.; Fan, C. Nucleic Acid Res. 2005, 33, e83. (19) Gaylord, B. S.; Massie, M. R.; Feinstein, S. C.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 34. (20) Tian, N.; Tang, Y.; Xu, Q.; Wang S. Macromol. Rapid Commun. 2007, 28, 729. (21) Ho, H. A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dore´, K.; Boudreau, D.; Leclerc, M. Angew. Chem., Int. Ed. 2002, 41, 1548. (22) Sehorn, M. G.; Sigurdsson, S.; Bussen, W.; Unger, V. M.; Sung, P. Nature 2004, 429, 433. (23) Li, Q.; Luan, G.; Guo, Q.; Liang, J. Nucleic Acid Res. 2002, 30, e5. (24) Xiao, Y.; Lubin, A. A.; Baker, B. R.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16677.

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displacement instead of a DNA hybridization event to attain high selectivity, thus offering a new strategy for DNA detection based on CPs. EXPERIMENTAL SECTION Materials and Measurements. DNA/RNA chimeric oligonucletide S labeled at the 5′-terminus with a fluorescein was obtained from TaKaRa Corporation. All of the other oligonucleotides were obtained from the Shanghai Sangon Biological Engineering Technology Service Co. Ltd. The concentrations of the DNA were obtained by measuring the absorbance at 260 nm. SYBR Gold nucleic acid stain was purchased from Invitrogen. Endonuclease Hae III was obtained from TaKaRa Corporation. Poly{(1,4-phenylene)-2,7-[9,9-bis(6′-N,N,N-trimethylammonium)hexyl fluorene]dibromide} (CCP-1) was synthesized according to the procedure in the literature. 25 UV-vis absorption spectra were taken on a JASCO V-550 spectrophotometer. Fluorescence measurements were obtained in a 3 mL quartz cuvette at room temperature using a Hitachi F-4500 fluorometer equipped with a xenon lamp excitation source. The excitation wavelength is 380 nm. Electrophoresis analysis experiments were performed as follows: the DNAs before and after cleavage by nucleases were loaded onto 15% or 18% nondenaturing polyacrylamide gel in 1× TBE buffer (8.9 mM Tris-base, 8.9 mM boric acid, 0.2 mM EDTA, pH 7.9) followed by electrophoresis at 110 V for 1.2 h. After the gel was stained by SYBR for 30 min, the photograph was taken in a WD-9403F UV device by a Canon digital camera. For the single-nucleotide mismatch assay, the gel was not stained and the photograph was directly taken. The water was purified using a Millipore filtration system. The water used for the DNAzyme assay was dealed with the 1% DEPC. DNA Detection Using CCP-1/P1 + P2/Hae III System. Amounts of 5.0 µL of P2 (10 µM) and 7.5 µL of P1 (10 µM) were annealed at 80°C for 20 min and then slowly cooled to room temperature to get the duplex. Then 7.5 µL of TC (10 µM), 2.5 µL of 10× buffer of Hae III and 1.5 µL of Hae III (10 unit/µL) were added into the duplex solution, and the final volume of the sample was adjusted to 25 µL by the Millipore water. After incubating at 37 °C for 50 min, 10 µL of the reaction solution were drawn out and added into 2.0 mL of HEPEs buffer (25 mM, pH ) 8.0) containing 1.5 µM CCP-1. The fluorescence spectra were measured at room temperature. In the case of DNA mismatch detection, the assays were performed in the same condition as above except for using target DNAs with one, two, three, and five bases mismatch (named as T1NC, T2NC, T3NC, and T5NC, respectively) instead of full matched TC. DNA Detection Using DNAzyme System. Amounts of 2.0 µL of P3 (10.0 µM) and 2.0 µL of P4 (10.0 µM) were annealed at 80 °C for 20 min and then slowly cooled to room temperature to get the duplex and then was diluted to 1 mL with HEPEs buffer (25 mM, pH ) 7.5) containing 50 mM MgCl2. To this solution were added 0.5 µL of S (10 µM) and 2 µL of MC (10 µM). After incubation at 37 °C for 1 h, 8 µL of CCP-1 (0.1 mM) and 3 µL of 10 µM P3 (to diminish the nonspecific interaction) were added. The fluorescence spectra were measured at room temperature. (25) Stork, M.; Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Adv. Mater. 2002, 14, 361.

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Scheme 1. (A,B) Schematic Representation of the DNA Detection, (C) Chemical Structures of CCP-1 and the DNAs

In the case of DNA mismatch detection, the assays were performed in the same condition as above except for using target DNAs with one, two, three, and five bases mismatch (named as M1NC, M2NC, M3NC, and M5NC, respectively) instead of full matched MC. RESULTS AND DISCUSSION Our new strategy for selective DNA detection is illustrated in Scheme 1A. The single-stranded P1 (35 mer) and P2 (43 mer) labeled at the 5′-terminus with a fluorescein (Fl) partly hybridize to form the P1/P2 duplex (27 bases match) that acts as the probe. P2 itself in the absence of P1 is a hairpin DNA with a 12 base pairs’ double-stranded stem and a 19 bases’ loop. The stem of P2 contains a recognition site (5′-d(GGCC)-3′) of endonuclease Hae III.26 When a complementary DNA (TC) of P1 is added into the P1/P2, the P2 is displaced from the P1/P2 duplex to form the more stable P1/TC duplex (32 bases match).23 The released P2 folds into a hairpin structure, and then the stem is cleaved upon adding Hae III to generate short DNA fragment carrying Fl. Upon adding the cationic conjugated polyelectrolyte CCP-1 (see Scheme 1C), the relatively weak electrostatic interactions between the DNA fragment and CCP-1 lead Fl far away from CCP-1 and inefficient fluorescence resonance energy transfer (FRET) between them is present.27 The ssDNA-Fl with less than six bases affords inefficient FRET from CCP-1 to fluorescein.27 When a (26) Blakesley, R. W.; Dodgson, J. B.; Nes, I. F.; Wells, R. D. J. Biol. Chem. 1977, 252, 7300. (27) Feng, F.; Tang, Y.; He, F.; Yu. M.; Duan, X.; Wang, S.; Li, Y.; Zhu, D. Adv. Mater. 2007, 19, 3490.

Figure 1. (a) Emission spectra and (b) intensity ratio of I424nm/I527nm from solutions containing CCP-1, P1/P2, and Hae III in the presence of TC or TNC in HEPEs buffer (25 mM, pH ) 8.0). The DNA reaction solutions are diluted by 200 times with HEPEs buffer before fluorescence measurements. The final amount: P1 ) 15 pmol, P2 ) 10 pmol, [Hae III] ) 3.0 unit/mL, TC or TNC ) 15 pmol, [CCP-1] ) 1.5 µM in RUs. (c) Emission spectra and (d) intensity ratio of I424nm/I527nm for CCP-1/P3 + P4/S in the presence of MC or MNC in HEPEs buffer solution (25 mM, pH ) 7.5) containing 50 mM MgCl2. P3 ) P4 ) 20 pmol, S ) 5 pmol, MC or MNC ) 20 pmol, [CCP-1] ) 0.8 µM in RUs. The excitation wavelength is 380 nm. Error bars were obtained from three independent measurements.

noncomplementary TNC is added, P2 is not displaced and no hairpin structure forms, thus the P2 cannot be cleaved by Hae III. In this case, upon adding CCP-1, the strong electrostatic interactions between P1/P2 and CCP-1 keep them in close proximity, allowing for efficient FRET from CCP-1 to Fl. To avoid utilizing unstable Hae III endonuclease, we design a new system based on RNA-cleaving DNAzyme.28,29 As shown in Scheme 1B, DNAzyme (P4) forms partly matched duplex (15 bases match) with P3 where its enzyme activity is inhibited. The S containing two RNA bases as cleavage site is the substrate of P4.9 When complementary MC of P3 is added into the P3/P4 duplex, the P4 is displaced to form the more stable P3/MC duplex (21 bases match). Then the released P4 binds with the S and cleave it to generate short DNA fragment carrying Fl. Upon adding CCP-1, inefficient FRET from CCP-1 to Fl is present. For noncomplementary MNC, P4 cannot be displaced and the S cannot be cleaved. At this case, upon adding CCP-1, efficient FRET from CCP-1 to Fl is present. Thus, the DNA can be detected by fluorescence spectra in view of the observed CCP-1 or Fl emission changes in aqueous solutions. Figure 1a compares the emission spectra observed upon adding CCP-1 ([CCP-1] ) 1.5 µM in repeat units (RUs)) to assay solutions containing P1/P2 ([P2] ) 10 pmol, [P1] ) 15 pmol) and Hae III (3.0 unit/mL) in the presence of TC or TNC ([TC] or [TNC] ) 15 pmol)in HEPEs buffer solution (25 mM, pH ) 8.0). The initial solution of CCP-1/P1 + P2/Hae III shows intense Fl emission at 527 nm resulting from the efficient FRET from CCP1. Upon adding complementary TC, the emission intensity of CCP-1 (28) Santoro, S. W.; Joyce, G. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4262. (29) Joyce, G. F. Annu. Rev. Biochem. 2004, 73, 791.

Figure 2. (a) Electrophoresis analysis of P1/P2 in the presence of target DNA cleaved by Hae III: lane 1 for P1/P2; lane 2 for P1 + TC; lane 3 for P2 only; lane 4 for P2 in the presence of Hae III; lane 5 for P1+P2/TC in the absence of Hae III; lane 6 for P1/P2 in the presence of Hae III; lane 7 for P1/P2/TC in the presence of Hae III; lane 8 for P1/P2/T5NC in the presence of Hae III. (b) Electrophoresis analysis of P3 + P4/S in the presence of target DNA: lane 1 for P4 only; lane 2 for S only; lane 3 for P4/S; lane 4 for P3 + P4/S; lane 5 for P3 + P4/S/MC. The gels were stained by SYBR, and the photograph was taken in a WD-9403F UV device by a Canon digital camera.

at 424 nm is increased and that of Fl at 527 nm is decreased. For the noncomplementary TNC, both the emission intensity of CCP-1 at 424 nm and that of Fl at 527 nm were not changed. The comparison of the ratio of emission intensity at 424 and 527 nm (I424nm/I527nm) shows approximately four times higher signal for complementary target TC relative to the noncomplementary TNC (Figure 1b). For CCP-1/P3 +P4/S system, similar results were obtained as those of CCP-1/P1 +P2/Hae III system (Figure 1c,d). The comparison of the ratio value of I424nm/I527nm also shows approximately three times higher signal for complementary target MC relative to the noncomplementary MNC (Figure 1d). Direct evidence to show the DNA displacements and cleavages of DNAs are provided by electrophoresis analysis using a 15% Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

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Figure 3. Emission spectra (a) and intensity ratio of I424nm/I527nm (b) for CCP-1/P1 + P2/Hae III as functions of the concentration of target DNA TC in HEPEs buffer solution (25 mM, pH ) 8.0). The DNA reaction solutions are diluted 200 times with HEPEs buffer before fluorescence measurements. The final amount: P1 ) 10 pmol, P2 ) 10 pmol, Hae III ) 3.0 unit, TC ) 0∼15 pmol, [CCP-1] ) 1.5 µM in RUs. (c) Emission spectra and (d) intensity ratio of I424nm/I527nm for CCP-1/P3 + P4/S as functions of the concentration of target DNA MC in HEPEs buffer solution (25 mM, pH ) 7.5) containing 50 mM MgCl2. P3 ) P4 ) 20 pmol, S ) 5 pmol, MC ) 0∼20 pmol, [CCP-1] ) 0.8 µM in RUs. The excitation wavelength is 380 nm. Error bars were obtained from three independent measurements.

Scheme 2. The Sequences of the Target DNAs, The TC Is a Strand That Is Perfectly Complementary to P2, the T1NC, T2NC, T3NC, and T5NC Are Strands That Are Complementary to P2 with One, Two, Three, and Five-Base Mismatches (Underlined), The MC Is a Strand That Is Perfectly Complementary to P4, the M1NC, M2NC, M3NC, and M5NC Are Strands That Are Complementary to P4 with One, Two, Three, and Five-Base Mismatches (Underlined).

III. In the presence of the noncomplementary TNC target, the P2 cannot be displaced and cleaved as shown in lane 8. For P3 + P4/S system, similar results were obtained as shown in Figure 2b. These results are well consistent with the FRET results as shown in Figure 1. Noted that with a standard commercial fluorometer (Hitachi F-4500) equipped with a xenon lamp excitation source and a photomultiplier tube, the limit of detection (LOD) of this method is obtained from eq 1 by 11 independent measurements:30

LOD ) 3 ×

nondenaturing polyacrylamide gel (Figure 2). As shown in Figure 2a, lane 5 for the P1 + P2/Tc mixture in the absence of Hae III moves as two bands, respectively, corresponding to the P1/Tc duplex and displaced P2, which indicates that the P2 is displaced from the P1/P2 probe duplex by P1’s complementary Tc strand to form the more stable P1/Tc duplex. Lane 6 for the P1/P2 duplex in the presence of Hae III moves as only one band, which shows that the Hae III cannot cleave the P2 in the P1/P2 duplex. Lane 7 for the P1/P2/Tc mixture in the presence of Hae III moves as two bands, respectively, corresponding to the P1/Tc duplex and cleaved fragments of P2, which indicates that the P2 is displaced from the P1/P2 probe duplex by Tc following the cleavage by Hae 2242

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S0 S

(1)

where S0 is the standard deviation of the background and S is the sensitivity. The detection limits of the target DNA are 2.14 pmol for the CCP-1/P1 + P2/Hae III system and 0.75 pmol for the CCP-1/P3 + P4/S system (Figure 3). These data nicely demonstrate the generality of Scheme 1A,B in providing platforms for sensitive fluorescence detection of DNA. Comparing parts b and d of Figure 3, the ratio changes of I424nm/I527nm for CCP-1/P1 + P2/Hae III changes less than those for CCP-1/P3 + P4/S and show that the latter approach is better for DNA detection. To evaluate the selectivity of our new DNA sensing platforms, the effect of base mismatch in the complementary strand on intensity ratio of I424nm/I527nm was studied in the presence of target DNAs with one, two, three, and five bases mismatch compared with complementary TC or MC (see Scheme 2 for sequences) under conditions similar to those in Figure 1 (Figure 4). As shown (30) Eggins, B. R. Chemical Sensors and Biosensors; John Wiley & Sons, Ltd: West Sussex, England, 2002.

Figure 4. (a) The ratio of I424nm/I527nm for CCP-1/P1 + P2/Hae III in HEPEs buffer (25 mM, pH ) 8.0) as a function of the number of mismatched bases in the complementary strand. The final amount: P1 ) 15 pmol, P2 ) 10 pmol, Hae III ) 3.0 unit, TC ) T1NC ) T2NC ) T3NC ) T5NC ) 15 pmol, [CCP-1] ) 1.5 µM in RUs; the excitation wavelength is 380 nm. (b) Electrophoresis analysis of P1 + P2/Hae III in the presence of target DNA: lanes 3-7 for DNAs with zero, one, two, three, and five-base mismatches in the complementary strand, respectively; lane 1 for P1/P2/TC without Hae III; lane 2 for P1/P2 with Hae III. (c) The intensity ratio of I424nm/I527nm for CCP-1/P3 + P4/S as a function of the number of mismatched bases in the complementary strand. P3 ) P4 ) 20 pmol, S ) 5 pmol, MC ) M1NC ) M2NC ) M3NC ) [M5NC] ) 20 pmol, [CCP-1] ) 0.8 µM in RUs; the excitation wavelength is 380 nm. Error bars were obtained from three independent measurements.

in Figure 4a, the ratio value of I424nm/I527nm follows the order DNA5NC ) DNA3NC < DNA1NC < DNAC. The similar results for CCP-1/P3 + P4/S system were obtained (Figure 4c). The gel image displayed in Figure 4b clearly proves the results of fluorescence experiment by monitoring the intensity of the bands of the P1/P2 duplex and the DNA fragment carrying Fl. The gel was not stained and the visible bands come from the fluorescence of Fl. It shows that the intensity of the P1/P2 band increases and that of DNA fragment decreases as the increase of mismatched bases in the target DNA. These results show that increasing the number of mismatch bases inhibits the displacement of P2 from the P1/P2 duplex. Thus the single-nucleotide mismatch can be clearly detected in target DNA in aqueous solutions. CONCLUSION In summary, a new methodology has been developed for DNA detection that interfaces target-induced DNA strand displacement

with the light harvesting properties of conjugated polyelectrolytes. Single-nucleotide mismatch can be clearly detected in target DNA. The protocol offers a convenient approach for homogeneous, selective and sensitive DNA assay in aqueous solution without using any denaturation steps. ACKNOWLEDGMENT The authors are grateful for the financial support from the “100 Talents” program of the Chinese Academy of Sciences, the National Natural Science Foundation of China (Grants 20725308, 20574073), and the National High-Tech R&D Program (Grant No. 2006AA02Z130).

Received for review November 25, 2007. Accepted December 21, 2007. AC702415P

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