J. Phys. Chem. B 2008, 112, 1783-1788
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Conformational Change Detection of DNA with the Fluorogenic Reagent of o-Phthalaldehyde-β-Mercaptoethanol Yun Fei Long,†,‡ Qie Gen Liao,† Cheng Zhi Huang,*,† Jian Ling,† and Yuan Fang Li† College of Chemistry and Chemical Engineering, Southwest UniVersity, CQKL-LRTA, Chongqing 400715, China, and Institute of Chemistry and Chemical Engineering, Hunan UniVersity of Science and Technology, Hunan 411201, China ReceiVed: February 27, 2007; In Final Form: NoVember 11, 2007
o-Phthalaldehyde-β-mercaptoethanol (OPAME) as a fluorogenic reagent has been found wide applications in the detection of amino acids based on its reaction with primary amino groups. In this contribution, we report our new findings concerning the reactions of OPAME with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), respectively. It has been found that ssDNA can react with OPAME easily as a result of giving rise to strong fluorescence emissions, while dsDNA, prepared by hybridizing ssDNA with its complementary target prior to the reaction, displays inert chemical activity and gives out weak fluorescence emission. Mechanism investigations have shown that the reaction activity between OPAME and DNA depends on the amino groups that are related to the conformation of uncoiled and exposed extent of DNA structure, and thus the inert chemical activity of dsDNA results from screening of the dsDNA bases in the interior of the double strands. Therefore, we could design a way to detect conformation change of DNA with OPAME and further develop a novel, simple label-free sequence detection method for complementary and single-base mismatched ssDNA in the hybridization of DNA.
Introduction Single-stranded DNA (ssDNA) probe has been widely applied in medical diagnostics and biomedical research in recent decades owing to its high binding affinity and high selectivity for targets such as complementary ssDNA, metal ions, small molecules, proteins, or even whole cells.1-6 Compared with rival antibodies or artificial receptors, ssDNA probe is ideal since it holds the advantages of simple synthesis, easy labeling, good stability, and wide applicability. Based on the conformational change of ssDNA probe upon binding target, fluorescence sensors for target detections have been proposed through modification with fluorophores, which, however, has the drawbacks of being timeconsuming and having complicated operation.3,7-11 In such cases, it is desirous to develop label-free methods with novel artificial fluorescent reagents such as polythiophene or [Ru(phen)2(dppz)]2+, which are generally based on the physical interactions such as electrostatic attraction or intercalated binding between the reagents and DNA.12-16 o-Phthalaldehyde-β-mercaptoethanol (OPAME) is a wellknown fluorogenic reagent for the analysis of amino compounds since it can easily react with primary amino compounds as a result of producing thio-substituted isoindole compounds with high quantum yield of fluorescence emission (Scheme S1 in the Supporting Information displays the principle of this reaction).17,18 To the best of our knowledge, it has been seldom applied to investigation of the properties of DNA. We found that ssDNA could react with OPAME easily as a consequence of giving rise to strong fluorescence emissions, and doublestranded DNA (dsDNA) has inert chemical activity with OPAME and displays weak fluorescence emission.19-21 Thus, * Corresponding author. E-mail:
[email protected]. † Southwest University. ‡ Hunan University of Science and Technology.
in this contribution, we develop a conformation-dependent method based on the fact that DNA has different reaction activities with OPAME, which are greatly different from those reports.7-16 Experimental Section Materials. ssDNAs were synthesized by Beijing Sunbiotechnology Co. (Beijing, China) and used without further purification. The used ssDNA sequences include probes (P) and targets (T). They are P1, 5′- ATC ACA GTT AAA TTG C -3′, its complementary target (T1), 5′- GCA ATT TAA CTG TGA T -3′, and its single-base mismatched target (MT), 5′- GCA ATT TAA GTG TGA T -3′. Natural and thermally denatured fish sperm DNA were used for comparison. Guanosine-5′monophosphoric acid (GMP), adenosine-5′-monophosphoric acid (AMP), cytosine, and thymine were employed to identify the reaction products for mechanism investigation. Both o-phthaldialdehyde (OPA) and β-mercaptoethanol (βME) were commercially purchased from Sigma. To obtain the OPAME working reagent (1.9 mM), 25 mg of OPA was first dissolved in 5 mL of HPLC-grade methanol (OPA-methanol) within 1 min with moderate agitation. Second, 10 µL of β-ME was added to the OPA-methanol solution in a fume cupboard and the mixture was agitated. Finally, the OPAME solution was diluted to 100 mL with borate buffer solution of pH 9.5 (80 mM of boracic acid was adjusted to pH 9.5 with 2.0 M NaOH), and the reagent was left to stand for at least 2 h (preferably overnight) to reduce the background fluorescence. Hybridization buffer solution containing 50 mM boracic acid and 0.5 M NaCl was adjusted to pH 8.0 with NaOH solution. All chemicals were analytical reagents and used without further purification. Milli-Q purified water (18.2 MΩ) was used for all sample preparations. Apparatus. Absorption and fluorescence measurements were made with a U-3010 spectrophotometer and F-4500 spectro-
10.1021/jp071601g CCC: $40.75 © 2008 American Chemical Society Published on Web 01/24/2008
1784 J. Phys. Chem. B, Vol. 112, No. 6, 2008
Long et al.
Figure 1. IR spectra of GMP (a), AMP (b), and cytosine (c) and their reaction products with OPAME, respectively.
fluorometer (Hitachi Ltd., Tokyo, Japan), respectively. CD spectra were measured with a J-810 spectropholarimeter (Jasco, Tokyo, Japan). Spectrumgx spectrophotometer (Perkin-Elmer, USA) was used for recording IR spectra. An API 2000 mass spectrometer (Perkin-Elmer, USA) was used for detecting the mass of the reaction products of OPAME with GMP, AMP, and cytosine. A pHS-3C digital pH meter (Leici, Shanghai) was used to detect the pH values of the aqueous solutions. A Nikon coolpix-4500 digital camera was used to detect the fluorescence images. A portable 253 nm UV lamp was used as a light source for the mismatch detection of DNA. A MVS-1 vortex mixer (Beide Scientific Instrumental Ltd., Beijing, China) was used to blend the reaction solutions. Experimental Procedures. To obtain the reaction products of OPAME with GMP, AMP, cytosine, and thymine, we mixed OPAME (1 mmol, 10 mL of 50% (H2O/EtOH, v/v)) with these amino reactants (1 mmol in 10 mL of 50% H2O/EtOH, v/v) and kept the reaction solution for 2 days at room temperature. After vaporization of part of the reaction solvent and filtering and drying of the depositions, we could obtain the products. As for hybridization detection, 20.0 µL of probe (P) DNA solution, 40.0 µL of hybridization buffer solution, and 20.0 µL of target (T) DNA solution were added into a 1.5 mL eppendorf cup, respectively. After vortexing and incubation for 20 min at
the temperature of 37 °C, 100.0 µL of 1.9 mM OPAME solution was added and mixed thoroughly. After standing for 5 min, the mixture was transferred for fluorescence measurements by scanning the emission monochromator from 370.0 to 700.0 nm with the excitation at 340.0 nm, and other measurements including UV-vis and CD. Results and Discussions Reaction of OPAME with the Amino Groups of DNA. To learn the reaction nature of OPAME and DNA, we first investigated the reaction properties of OPAME with AMP, GMP, cytosine, and thymine, respectively. Figure 1 displays the involved IR spectra. It could be seen that the stretching vibrations of amino group at 3499 and 3416 cm-1 for GMP, 3364 and 3173 cm-1 for AMP, and 3171 and 3382 cm-1 for cytosine disappear after they react with OPAME, while the carbonyl stretching vibration (V) of substituted benzene comes into being at 762 cm-1 for GMP-OPAME product, 754 cm-1 for AMP-OPAME product, and 760 cm-1 for cytosineOPAME product. These phenomena indicate that the thiosubstituted isoindole compounds have been formed after OPAME reacts with GMP, AMP, and cytosine, respectively. Figure 2 shows that the resulting products have the molecular fragments
Conformational Change Detection of DNA with OPAME
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Figure 2. MS spectra of products of OPAME respectively with GMP (a), AMP (b), and cytosine (c).
of m/z 537.0 for GMP, 521.0 for AMP, and 306 for cytosine, which are identical to the practical m/z values of thio-substituted isoindole compound including m/z 539.0 for GMP, 523.0 for AMP, and 305 for cytosine. Therefore, we have reasons to deduce that thio-substituted isoindole compounds have been formed (Scheme S2 in the Supporting Information) for the reaction of OPAME with AMP, GMP, and cytosine, respectively.22,23 Nonetheless, similar results could not be found for the reaction of thymine with OPAME. We suppose that thymine only has a secondary amino group and could not effectively form thio-substituted isoindole compounds with OPAME.24,25 Thus, we can conclude that the reaction between OPAME with DNA exists in the primary amino group of DNA base as a result of forming the thio-substituted isoindole compound.
Chemical Reaction Activities of DNA with OPAME. It was found that the fluorescence emission of free OPAME is weak at 455 nm when excited at 340 nm (curve 1 in Figure 3), but could be enhanced about 6-fold by ssDNA, such as P1 (curve 3 in Figure 3) or T1 (not shown), indicating that the amino groups of ssDNA can react easily with OPAME as a consequence of forming strong fluorescent products. However, if ssDNA probe (P1) was hybridized first with its complete complement ssDNA target (T1), forming DNA hybrid (P1T1), then the reaction between the P1-T1 hybrid with OPAME are limited (curve 2 in Figure 3). Similar phenomena could be observed from the reaction of OPAME with natural and thermally denatured fsDNA, respectively (Figure S1 in the Supporting Information), showing that the chemical reaction
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Figure 3. Fluorescent excitations (left) and emissions (right) spectra of OPAME and its bindings with DNA. 1, OPAME (black); 2, OPAME-P1-T1 (red); 3, OPAME-P1 (green); 4, OPAME-P1-MT (blue). Conditions: cOPAME, 1.0 × 10-3 M; cP1, cT1, and cMT, 6.0 × 10-7 M. The inserted plot shows the fluorescence emission images, which were obtained by dropping 100 µL of solution on the surface of glass slides and recorded using a Nikon coolpix-4500 digital camera under the excitation of a portable 253 nm UV lamp light source.
Figure 4. Absorption spectra of OPAME and its mixtures with DNA. 1, P1 (black); 2, P1-T1 (gray green, mixture after hybridization); 3, P1-T1 (red, mixture immediately); 4, OPAME (violet); 5, OPAMEP1 (green); 6, OPAME-P1-T1 (aubergine); 7, OPAME-P1 (blue). Conditions: cOPAME, 1.0 × 10-3 M; cP1 (1, 2, 3, 5, 6, 1.5 × 10-6 M; 7, 3.0 × 10-6 M), cT1, 1.5 × 10-6 M.
activity of ssDNA with OPAME is much higher than that of dsDNA with OPAME. To manifest the dependence of the reaction on the structure of DNA, we can further measure the UV-vis and CD spectra of the reaction and the changes of fluorescence emissions with the temperature. It was known that the hybridization of DNA depends on the environmental conditions including incubation temperature and time. Figure 4 shows that the absorbance of the mixture between P1 and T1 at 260 nm decreases about 11% after incubation for 10 min at 37 °C (curves 2 and 3 in Figure 4), indicating that stable double-helix geometry of DNA can be formed under the incubation conditions.26 On the other hand, the absorption of P1 (curve 1 in Figure 4), T1 (not shown), and OPAME is very weak at 340.0 nm, but increases if P1 or T1 is
Long et al.
Figure 5. CD spectra of P1 and P1-T1 and their mixtures with OPAME. 1, P1 (black); 2, P1-OPAME (red); 3, P1-T1-OPAME (green); 4, P1-T1 (blue). Conditions: cOPAME, 1.0 × 10-3 M; cP1 and cT1, 1.5 × 10-6 M.
mixed with OPAME, illustrating that new complexes have formed concerning the reaction of OPAME with P1 or T1. In addition, it has been found that the absorbance at 340.0 nm increases with increasing P1 concentration (curve 7 in Figure 4). However, the absorbance of P1-T1 (curve 4 in Figure 4) at 340.0 nm becomes enhanced slightly in the presence of OPAME, and the absorbance of P1-T1-OPAME is much less than that of P1-OPAME at 340.0 nm (curves 5 and 6 in Figure 4), even though the total concentration of P1 and T1 is twice as that of P1, indicating that the chemical reaction activity of ssDNA with OPAME is higher than that of dsDNA. The CD spectra of P1 exhibit a positive Cotton effect at 278 nm and a weak negative Cotton effect at 249 nm (curve 1 in Figure 5), corresponding to the base stacking and the helicity of the DNA conformation, respectively.27 In contrast, P1-T1 exhibits a strong negative Cotton effect at 248.7 nm and a relative weak positive Cotton effect at about 280.9 nm compared with its negative Cotton effect (curve 4 in Figure 5), suggesting that P1-T1 is some canonical B-DNA with the stable doublehelix geometry structure.27,28 Upon binding with OPAME, the positive Cotton effect at 278 nm and negative Cotton effect of P1 at 249 nm are blue-shifted to 270.1 and 248.6 nm, respectively (curve 2 in Figure 5), while those of P1-T1 are not obviously shifted (curve 3 in Figure 5). On the other hand, the similarity of CD spectra between P1-T1 and P1-T1OPAME is greater than that between P1 and P1-OPAME over the wavelength range of 225.0-300.0 nm. These phenomena imply that the chemical reaction activity of ssDNA is much higher than that of dsDNA. The reaction activity of DNA with OPAME could be further identified by the dependence of fluorescence emission of OPAME-P1 and P1-T1-OPAME on temperature (Figure 6). It can be seen that the fluorescence emissions of OPAME scarcely change with increasing temperature over the range of 30-80 °C. While that of OPAME-P1 decreases quickly at first because of the light blanching effect, it decreases slowly after the temperature reaches about 38 °C where some coiling structures of P1 may be destroyed and the amino groups are exposed, indicating that the exposed amino groups have reaction activity with OPAME. As for P1-T1-OPAME, although the variation of fluorescence emissions is similar to that of OPAME-P1, the turning temperature should reach about 42 °C, and after that the fluorescence emission gradually recovers with the increasing temperature. The reason may be
Conformational Change Detection of DNA with OPAME
Figure 6. Fluorescence emissions of OPAME reacting products with ssDNA and dsDNA at different temperatures. 1, OPAME (black); 2, OPAME-P1-T1 (red); 3, OPAME-P1 (green). Conditions: cOPAME, 1.0 × 10-3 M; cP1 and cT1, 6.0 × 10-7 M; pH 9.5.
Figure 7. Kinetics of OPAME reaction with ssDNA and dsDNA. 1, OPAME (black); 2, OPAME-P1-T1 (red); 3, OPAME-P1 (green); 4, OPAME-P1-MT (blue). Conditions: cOPAME, 1 × 10-3 M; cP1 and cT1, 6.0 × 10-7 M; pH 9.5.
that P1-T1 hybrid is decomposed into single-stranded state at the temperature of 42 °C, and many more amino groups of DNA are exposed gradually with the increasing temperature, making it much easier for DNA to react with OPAME, resulting in stronger fluorescence emission. Reaction Kinetics of DNA with OPAME. To understand the effect of DNA conformation on the reaction, we tested the reaction kinetics of DNA with OPAME (Figure 7). It can be seen that the fluorescence emission of OPAME remains stable (curve 1 in Figure 7) in the absence of DNA within 10 min, while that of OPAME-P1 (curve 3 in Figure 7) becomes stronger and stronger and reaches maximum at about 2 min, and then decreases with further reaction progress caused by the effect of light blanching. Similar phenomena could be observed in the system of OPAME-P1-MT (curve 4 in Figure 7), but it reaches the maximum at about 2.5 min. The fluorescence emission of OPAME-P1-T1 (curve 2 in Figure 7), however, is very weak compared with that of OPAME-P1, and the weak enhancement of fluorescence emission reaches maximum at about 1.5 min, and after that, it scarcely decreases with the time scan. According to the above phenomena and the fact that the base pairs of dsDNA could be open in solution,29 we can deduce that the open speed of base pairs of P1-MT is quicker than that of P1-T1. This investigation on the reaction kinetics shows
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Figure 8. Reaction kinetics of P1-T1 with formaldehyde and OPAME, respectively. Reaction kinetics of P1-T1 with formaldehyde (1, black) and OPAME (2, red). Conditions: cOPAME, cformaldehyde, 1.0 × 10-3 M; cP1-T1, 6.0 × 10-7 M; λ ) 260.0 nm.
that the reaction of OPAME with ssDNA is quick and that with dsDNA is slow. To compare the similarities between the reaction of OPAME with DNA and the very well studied reaction of formaldehyde with DNA,29-31 we have tested the absorbance changes of the reaction systems with the reaction time at 260.0 nm (Figure 8). The results showed that P1-T1 is completely opened at about 0.5 min in the presence of formaldehyde, while it is not completely opened even at 10 min in the presence of OPAME, indicating that the reaction speed of P1-T1 with OPAME is slower than that with formaldehyde since the steric barrier of OPAME is stronger than that of formaldehyde. On the other hand, the absorbance of the P1-T1-formaldehyde system is decreased gradually after 0.5 min; this may be related to the cross-linking reaction property of formaldehyde.30,31 Hybridization and Mismatch Detection. Curves 2 and 4 in Figure 3 show that the fluorescence signals of single-base mismatched DNA hybrid and OPAME mixture (P1-MTOPAME) are higher than that of the completely complementary DNA hybrid and OPAME mixture (P1-T1-OPAME). That should be ascribed to the incomplete hybridization of ssDNA with its mismatched target DNA, which could be the reason for the ease of reaction with OPAME since much of the free ssDNAs are kept in the solution, and the amino group of the mismatched DNA hybrid at the mismatch site could react with OPAME because of its noncompact structure.32 To intuitively observe the dependence of the fluorescence emission on the ssDNA sequences, we employed a portable 253 nm UV lamp as a light source to irradiate the solutions and recorded the images with a digital camera (the inserted graph of Figure 3). It could be obviously seen that the fluorescence of P1-T1-OPAME is weaker than that of P1-MT-OPAME. That is to say, we can identify the complementary and singlebase mismatched hybrid visually with this simple method. Conclusion In summary, we have demonstrated that the amino groups of GMP, AMP, cytosine, and ssDNA could react with OPAME easily, and the reaction activity depends on the state of amino groups of uncoiled or exposed DNA. When ssDNA binds with the target, the ligand-dependent fluorescence change is large enough and could be applied to detect the targets in homogeneous solution. This method depends on chemical reaction and is greatly different from the previous reports based on molecular recognition, in which physical interactions are generally
1788 J. Phys. Chem. B, Vol. 112, No. 6, 2008 involved.33-38 We believe that OPAME has promise of avoiding false positive or negative results for analyte by a DNA-based solid-state detection device, and it has potential application for detecting aptamer-binding targets. Acknowledgment. We are thankful for the support of the National Natural Science Foundation of China (NSFC, NO. 20425517). Supporting Information Available: Experimental details and results. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Wang, L.; Yang, C. J.; Medley, C. D.; Benner, S. A.; Tan, W. H. J. Am. Chem. Soc. 2005, 127, 15664. (2) Long, Y. F.; Huang, C. Z.; Li, Y. F. J. Phys. Chem. B 2007, 111, 4535. (3) Ueyama, H.; Takagi, M.; Takenaka, S. J. Am. Chem. Soc. 2002, 124, 1428. (4) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J. Am. Chem. Soc. 2000, 122, 11547. (5) Zhang, H. Q.; Wang, Z. W.; Li, X. F.; Le, X. C. Angew. Chem., Int. Ed. 2006, 45, 1576. (6) Hermann, T. D.; Patel, J. Science 2000, 287, 820. (7) Merino, E. J.; Weeks, K. M. J. Am. Chem. Soc. 2003, 125, 12370. (8) Merino, E. J.; Weeks, K. M. J. Am. Chem. Soc. 2005, 127, 1276. (9) Nutiu, R.; Li, Y. Angew. Chem., Int. Ed. 2005, 44, 5464. (10) Rupcich, N.; Nutiu, R.; Li, Y.; Brennan, J. D. Anal. Chem. 2005, 77, 4300. (11) Rupcich, N.; Nutiu, R.; Li, Y. Brennan, J. D. Angew. Chem., Int. Ed. 2006, 45, 3295. (12) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954. (13) Ho, H.-A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384. (14) Liu, B.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 589. (15) Ho, H.-A.; Bera-Aberem, M.; Leclerc, M. Chem.-Eur. J. 2005, 11, 1718.
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