Protein Binding by a Molecular Light Switch Complex

Anal. Chem. 2004, 76, 5230-5235

Signaling Aptamer/Protein Binding by a Molecular Light Switch Complex Yaxin Jiang, Xiaohong Fang,* and Chunli Bai*

Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080 (China)

A novel method of signaling aptamer/protein binding for aptamer-based protein detection has been developed using a molecular light switch complex, [Ru(phen)2(dppz)]2+. The method takes advantage of the sensitive luminescence signal change of [Ru(phen)2(dppz)]2+ intercalating to the aptamer upon protein/aptamer binding. A 37-nt DNA aptamer against immunoglobulin E (IgE) was first tested as a model system. The luminescence of the [Ru(phen)2(dppz)]2+/IgE aptamer decreased with the increase of IgE. By monitoring the luminescence change, we were able to detect the binding events between the aptamer and IgE for IgE quantitation in homogeneous solutions as well as in serum. The assay was highly selective and sensitive with a detection limit of 100 pM for IgE. This new method is very simple and without the need for the covalent coupling of fluorophores to aptamers. The generalizability of the method was demonstrated by the direct detection of two other proteins, oncoprotein platelet derived growth factor-BB (PDGF-BB) using its DNA aptamer and r-thrombin using its RNA aptamer. This new approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies. There have been recent advances in the in vitro selection of nucleic acid aptamers for their specific binding to a variety of target molecules and in the exploitation of their potential in bioanalytical and biotechnological applications.1,2 Among them, the development and application of protein aptamers is of particular interest. Due to their high binding affinity, simple synthesis, easy storage, and wide applicability, aptamers are emerging as a new class of molecules that rival commonly used antibodies in protein recognition, sensing, and profiling. The key in the development of aptamer-based analytical methods and sensors is to transduce aptamer recognition events to detectable signals.3-11 “Signaling aptamers” having the ability * Corresponding authors. E-mail: [email protected] (X.F.); clbai@ iccas.ac.cn (C.B.). (1) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818-822; Tuerk, C.; Gold, L. Science 1990, 249, 505-510. (2) Gold, L.; Polisky, B.; Uhlenbeck, O.; Yarus, M. Annu. Rev. Biochem. 1995, 64, 763-97; Hesselberth, J.; Robertson, M. P.; Jhaveri, S.; Ellington, A. D. Rev. Mol. Biotechnol. 2000, 74, 15-25. (3) Jhaveri, S.; Kirby, R.; Conrad, R.; Maglott, E. J.; Browser, M.; Kennedy, R. T.; Glick, G.; Ellington, A. D. J. Am. Chem. Soc. 2000, 122, 2469-2473. (4) Ellington, A. D.; Hieftje, G. M. Anal. Chem. 1998, 70, 3419-3425. (5) Fang, X.; Cao, Z.; Beck, T.; Tan, W. Anal. Chem. 2001, 73, 5752-5757.

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to directly report target concentration are actively being sought, and most signaling aptamers developed thus far are for homogeneous or heterogeneous fluorescence assay.3-9 For example, single-fluorophore-labeled aptamers can be used to signal binding by monitoring the changes of fluorescence intensity or anisotropy resulting from the changes of the microenvironment or rotational motion of the fluorophore.3-5 Taking advantage of the ligandinduced conformational change of aptamers, double-labeled aptamers have been developed to give target-dependent fluorescence changes through fluorescence energy transfer.6,7 However, as the precise target binding sites and the conformational changes of the aptamers are generally unknown, it is not easy to design labeling strategies. Besides, there is always a concern that the conjugation of a fluorophore to an aptamer will ultimately weaken the affinity of the aptamer to its ligand.8,9 Moreover, most of the signaling aptamers developed so far are labeled DNA aptamers, but the fluorescent labeling of RNA aptamers, which have a larger population than DNA aptamers, is difficult because of the instability of RNA molecules. Therefore, efforts have been made to develop alternative methods without aptamer labeling.8,9 Few simple and general strategies for aptamer-based protein detection are presently available. We report, however, a new method to signal aptamer/protein binding using [Ru(phen)2(dppz)]2+ (phen ) 1,10-phenanthroline, dppz ) dipyrido[3,2-a:2′,3′-c]phenazine, Figure 1A), a newly developed “light switch” complex.12,13 [Ru(phen)2(dppz)]2+ has no luminescence in aqueous solution as the triplet MLCT (metal-to(6) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J. Am. Chem. Soc. 2000, 122, 11547-11548; Stojanovic, M. N.; de Prada, P.; Landry, D. W. J. Am. Chem. Soc. 2001, 123, 4928-4931; Yamamoto, R.; Kumar, P. K. Genes Cells 2000, 5, 389-396. (7) Li, J.; Fang, X.; Tan, W. Biochem. Biophys. Res. Commun. 2002, 292, 3140. (8) Stojanovic, M. N.; Landry, D. W. J. Am. Chem. Soc. 2002, 124, 96789679; Wu, L. H.; Curran, J. F. Nucleic Acids Res. 1999, 27, 1512-1516; Jhaveri, S.; Rajendran, M.; Ellington, A. D. Nat. Biotechnol. 2000, 18, 12931297. (9) Nutiu, R.; Li, Y. F. J. Am. Chem. Soc. 2003, 125, 4771-4778; Ho, H. A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384-1387. (10) Fahlman, R. P.; Sen, D. J. Am. Chem. Soc. 2001, 124, 4610-4616; Seetharaman, S.; Zivarts, M.; Sudarsan, N.; Breaker, R. R. Nat. Biotechnol. 2001, 19, 336-341. (11) German, I.; Buchanan, D. D.; Kennedy, R. T. Anal. Chem. 1998, 70, 45404545; Liss, M.; Petersen, B.; Wolf, H.; Prohaska, E. Anal. Chem. 2002, 74, 4488-4495; Strouse, R. J.; Anderson, D. W.; Argentieri, D. C. J. Immunoassay 1991, 12, 113-24. (12) Turro, C.; Bossmann, S. H.; Jenkins, Y.; Barton, J. K.; Torro, N. J. J. Am. Chem. Soc. 1995, 117, 9026-9032; Jekins, Y.; Friedman, A. E.; Turro, N. J.; Barton, J. K. Biochemistry 1992, 31, 10809-10816; Holmlin, R. E.; Stemp, E. D.; Barton, J. K. Inorg. Chem. 1998, 37, 29-34. 10.1021/ac049565u CCC: $27.50

© 2004 American Chemical Society Published on Web 08/03/2004

Figure 1. Chemical structure of the “light switch” complex [Ru(phen)2(dppz)]2+ (A) and the estimated secondary structures of IgE DNA aptamer (B), PDGF-BB DNA aptamer (C), and R-thrombin RNA aptamer (D).

ligand charge transfer) excited state is effectively quenched by hydrogen binding between water and the phenazine nitrogen of the ligand. When it binds to dsDNA, the interaction between the ligand and the base pairs of duplex nucleic acid protects the phenazine nitrogen from water, leading to intense emission. [Ru(phen)2(dppz)]2+ has a high binding affinity to duplex nucleic acid (Ka: ∼106 M-1), and its luminescence intensity can be enhanced 104-fold.12,13 It has been widely used as a promising luminescent probe in DNA detection and structure analysis.12-14 Although aptamers are ssDNA or ssRNA, they usually fold into unique threedimensional structures through base pairing to ensure their specific binding to targets.6-10 The folded structures of the aptamers allow the intercalation of [Ru(phen)2(dppz)]2+ to emit luminescence. As the luminescence signature of [Ru(phen)2(dppz)]2+ is remarkably sensitive to its local environment and DNA/RNA conformation, and the binding of protein/aptamer usually changes or distorts the secondary structure of the aptamer,15,16 we hypothesized that upon protein binding the induced aptamer conformational change as well as the blocking of [Ru(phen)2(dppz)]2+ intercalation by protein would result in a significant protein-dependent luminescence change. (13) Hiort, C.; Lincoln, P.; Norde´n, B. J. Am. Chem. Soc. 1993, 115, 34483454; Lincoln, P.; Broo, A.; Norde´n, B. J. Am. Chem. Soc. 1996, 118, 26442653. (14) Ling, L. S.; Fang, X. H.; Wang, C.; Wan, L. J.; Chen, D. M.; Bai, C. L. Chem. Lett. 2003, 32, 80-81; Ling, L. S.; Song, G. W.; He, Z. K.; Liu, H. Z.; Zeng, Y. E. Microchem. J. 1999, 63, 356-364. (15) Werner, M. H., Gronenborn, A. M.; Clore, G. M. Science 1996, 271, 778784. (16) Wiegand, T. W.; Williams, P. B.; Dreskin, S. C.; Jouvin, M. H.; Kinet, J. P.; Tasset, D. J. Immunol. 1996, 157, 221-230.

To test the feasibility of this strategy, we selected three existing aptamers (structures shown in Figure 1B-D) against three disease-related proteins which are of importance in biomedical study: IgE, PDGF-BB, and thrombin. A 37-nt IgE aptamer11,16,17 was first used to construct the IgE signaling probe and was tested in detail. The results showed that the probe was able to detect IgE with high sensitivity and selectivity, even in a complex biological sample such as serum. The method also worked well for the detection of the oncoprotein PDGF-BB,5,18 demonstrating its potential to be a general method. R-Thrombin and its aptamer19 were chosen as a model system to examine the applicability of the strategy to RNA aptamers. The successful development of signaling RNA aptamers further suggested the broad applications of the method. To the best of our knowledge, this is the first report of using label-free DNA and RNA aptamers for homogeneous protein detection. EXPERIMENTAL SECTION Materials. Immunoglobulin E, purified from human plasma, was purchased from Athens Research & Technology Inc. (Athens, GA). Human serum, PDGF-BB, immunoglobulin M, immunoglobulin G, bovine serum albumin (BSA), lysozyme, cytochrome C, (17) Jiang, Y. X.; Zhu, C. F.; Ling, L. S.; Wan, L. J.; Fang, X. H.; Bai, C. L. Anal. Chem. 2003, 75, 2112-2116; Jiang, Y. X.; Wang, J.; Fang, X. H.; Bai, C. L. J. Nanosci. Nanotechnol., in press. (18) Green, L. S.; Jellinek, D.; Jenison, R.; O ¨ stman, A.; Heldin, C. K.; Janjic, N. Biochemistry 1996, 35, 14413-11424; Floege, J.; Ostendorf, T.; Janssen, U.; Burg, M.; Radeke, H. H.; Vargeese, C.; Gill, S. C.; Green, L. S. N.; Janjic Am. J. Pathol. 1999, 154, 169-179. (19) Kubik, M. F.; Stephens, A. W.; Schneider, D.; Marlar, R. A.; Tasset, D. Nucleic Acids Res. 1994, 22, 2619-2626.

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and hemoglobin were purchased from Sigma (St. Louis, MO). R-Thrombin was purchased from Haematologic Technologies Inc. (Essex Junction, VT). The R-thrombin RNA aptamer, 5′-GAGCA UGCUG GUGCG GCUUU GGGCG CCGUG CUU-3′, was custom synthesized from Integrated DNA Technologies, Inc. (Coralville, IA). All the DNA oligos were custom synthesized from SinoAmerica Bioengineering Company (Beijing, China). These include the 37-nt IgE DNA aptamer (AP1), 5′-GGGGC ACGTT TATCC GTCCC TCCTA GTGGC GTGCCCC-3′; the oligo which is complementary to AP1 (CAP1), 5′-GGGGCAC GCCAC TAGGA GGGAC GGATA AACGT GCCCC-3′; the polydeoxythymidine (dT37), 5′-TTTTT TTTTT TTTTT TTTTT TTTTT TTTTT TTTTTTT3′; the scramble oligos (C2) which had the same composition as AP1 but with a different base sequence, 5′-TTTTC CGACC TTCCG GGGGC CCCAG CGTCC TGCAG TG-3′; and the PDGF-BB DNA aptamer, 5′-CAGGC TACGG CACGT AGAGC ATCAC CATGA TCCTG-3′. [Ru(phen)2(dppz)]2+ was synthesized as in ref 14. TO and TOTO were purchased from Molecular Probes Inc. (Eugene, OR). Milli-Q purified water (18.2 MΩ) was used for all sample preparations. Unless specified, the buffers used were PBS buffer (20 mM Na2HPO4, pH ) 7.0) for IgE detection, the physiological buffer (20 mM Tris-HCl buffer with 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, pH ) 7.4) for PDGF-BB, and Tris buffer (50 mM Tris-HCl, 100mM NaCl, 1mM DTT, 1 mM MgCl2, pH ) 7.7) for R-thrombin. Instrumentation. All luminescence measurements were performed on a Fluorolog 3-21 spectrofluorometer (Jobin Yvon, Inc., Edison, NJ), except the luminescence lifetime that was measured on an NAES-1100 time-resolved spectrofluorometer (HORIBA, Japan). The sample cell was a 1-mL cuvette. For [Ru(phen)2(dppz)]2+, the excitation and emission wavelengths were 450 and 610 nm. Both excitation and emission slits were set at 7 nm in the experiments unless specified. The results were reported as mean values of triplicates. RESULTS AND DISCUSSION Signaling the Binding of the IgE/DNA Aptamer. Because protein IgE plays a key role in allergic responses, the development of its aptamers has attracted considerable research interest for both therapeutic and analytical purposes.16,17 The aptamer-based capillary electrophoresis (CE) method and quartz crystal microbalance (QCM) biosensor for IgE detection have been reported.11 Here, we selected the existing DNA aptamer (Figure 1B) for protein IgE to test the feasibility of our new strategy using the molecular light switch. As shown in Figure 2, [Ru(phen)2(dppz)]2+ alone has very low luminescence in solution when excited at 450 nm. Its luminescence intensity at 610 nm increased approximately 20 times with the addition of the 37-nt IgE DNA aptamer (the molar ratio of [Ru(phen)2(dppz)]2+/DNA was 8:1 to ensure the saturation of the intercalating dye and the maximum luminescence increase), while in the control experiment it showed only a weak luminescence with the addition of a linear 37-nt polydeoxythymidine. This result indicated that [Ru(phen)2(dppz)]2+ did intercalate into the aptamer, as the aptamer has a predicted stem-loop structure with nine Watson-Crick base pairs and three non-Watson-Crick base pairs in its stem.16 When the protein IgE was added to the luminescent aptamer/[Ru(phen)2(dppz)]2+ solution, a quick and significant luminescence intensity 5232 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

Figure 2. Luminescence emission spectra of (a) the IgE aptamer; (b) [Ru(phen)2(dppz)]2+; (c) mixture of the aptamer and [Ru(phen)2 (dppz)]2+; (d) mixture of the aptamer, Ru(phen)2(dppz)]2+, and IgE; (e) mixture of (dT)37 and [Ru(phen)2(dppz)]2+; (f) mixture of (dT)37, Ru(phen)2(dppz)]2+, and IgE. The concentration of the aptamer, Ru(phen)2(dppz)]2+, IgE, and (dT)37 were 5 × 10-9 M, 4 × 10-8 M, 2 × 10-8 M, and 5 × 10-9 M, respectively.

decrease was observed. The reaction reached equilibrium in about 3 min. Several other control experiments have been performed to confirm that the luminescence change is caused by the protein binding (see the Supporting Information). For example, IgE was added to a mixture of [Ru(phen)2(dppz)]2+ and the duplex DNA formed by the IgE DNA aptamer and its complementary ssDNA. Although the luminescence of the [Ru(phen)2(dppz)]2+/dsDNA was high, its signal was essentially the same in the absence or presence of IgE since the protein did not bind to the dsDNA. Detecting IgE with High Selectivity and Sensitivity. Due to the inherent specificity of the aptamer toward its target protein, the luminescence change of the aptamer/[Ru(phen)2(dppz)]2+ is highly selective. A fixed amount of the aptamer/ [Ru(phen)2(dppz)]2+ was incubated with either IgE or some other proteins such as BSA, hemoglobin, lysozyme, cytochrome C, myoglobin, IgG, and IgM. Only IgE caused a marked reduction in luminescence, while all the other proteins tested, even at 10-fold higher concentrations than those used for IgE, failed to cause significant change in the luminescence (Figure 3). Titration experiments were carried out by adding increasing amounts of IgE to aptamer/[Ru(phen)2(dppz)]2+ to examine whether the luminescence change could be used for IgE quantitation. Figure 4 shows that the luminescence intensity continued to decrease following the increase of the IgE concentration until a plateau (about 35% decrease) was reached. There was a good linear relationship between the luminescence change and the IgE concentration in the subnanomolar range of protein concentration for sensitive IgE quantitation when 5 nM of aptamer was used (with a correlation coefficient of 0.991 for the linear calibration curve shown in the inset of Figure 4). The detection limit for IgE was 100 pM experimentally determined based on a signal/noise > 3. This sensitivity is comparable or better than that of other reported aptamer-based analytical methods for IgE detection, such as 46 pM with CE and 500 pM with QCM, as well as the reported antibody-based ELISA (about 45 pM).11

Figure 4. Titration of the [Ru(phen)2(dppz)]2+/aptamer with IgE in the concentration range of 0-70 nM. Inset: the linear relationship of the luminescence change of the [Ru(phen)2(dppz)]2+/aptamer at 610 nm and the IgE concentration in the subnanomolar range (the y-intercept and slope of the linear curve were 0.0068 ( 0.0041 and 0.17 ( 0.0090, respectively). The concentrations of the aptamer and [Ru(phen)2(dppz)]2+ were 5 × 10-9 M and 4 × 10-8 M.

Figure 3. Different proteins were compared with IgE in their capability to change the luminescence intensity of the aptamer/[Ru(phen)2(dppz)]2+; 5 × 10-9 M IgE aptamer and 4 × 10-8 M [Ru(phen)2(dppz)]2+ were used. (A) The concentration of IgE was 1 × 10-9 M, while the concentrations of the other proteins were 1 × 10-8 M. (B) The concentrations of IgE, IgG, and IgM were 1 × 10-8 M, 1 × 10-7 M, and 1 × 10-7 M, respectively.

Our new solution-based method is much simpler with no need for the labeling or immobilization of either the aptamer or the protein. It was found that the presence of 100 nM serum albumin or 10 nM IgG did not affect the IgE subnanomolar detection limit. This indicates that a high sensitivity of protein detection can be achieved in complex biological samples. We further tried IgE detection in human serum, one of the most challenging media containing a variety of proteins including different types of immunoglobulins. As shown in Figure 5, the IgE titration curve in the 1% serum was similar to that in the PBS solution. The same detection limit as that in the PBS buffer was achieved with 50 nM aptamer. It is noteworthy that if a more concentrated serum was used, the background signal of the serum at 610 nm increased, hindering the sensitive monitoring of the [Ru(phen)2(dppz)]2+ luminescence change (see the Supporting Information). The higher background is caused by tailing of the broad serum peak around 520 nm (ex: 450 nm). The matrix interference on the IgE detection most likely resulted from background Raman scattering and fluorescent components in the serum, instead of cross-reaction with other proteins. Subnanomolar IgE detection was also achieved in the presence of 10 nM dsDNA (formed by the IgE DNA aptamer and its complementary ssDNA) when more

Figure 5. Titration of the [Ru(phen)2(dppz)]2+/aptamer with IgE in 1% serum in the concentration range of 0-50 nM. The concentrations of the aptamer and [Ru(phen)2(dppz)]2+ were 5 × 10-8 M and 4 × 10-7 M, respectively.

[Ru(phen)2(dppz)]2+ was used to ensure the saturation of the intercalating dye to the aptamer, further demonstrating the high selectivity of the method. Effect of pH and Ionic Strength of the Buffer. Some factors that affect protein/DNA interaction, such as the pH and ionic strength of the buffer, have been studied in IgE detection. The pH of the buffer we used in the above IgE detection (pH 7) was chosen according to that reported for the SELEX selection of the IgE aptamer.11 We expect that the pH is favorable to the binding of IgE to the aptamer and thus the protein detection. It is found that a change of pH in the range of 7-9, where the DNA aptamer is expected to be stable, did not have a strong effect on the sensitivity of IgE detection. There was a small increase in the IgE detection limit as the pH increased, e.g., the detection limit of IgE was 300 pM in the PBS buffer at pH 9 compared to 100 pM at pH 7. Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

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Table 1. Luminescence Lifetime of [Ru(phen)2(dppz)]2+

(S ) 5 ×

10-7

sample M aptamer + 4 × 10-6 M [Ru(phen)2(dppz)]2+)

S S + 2.5 × 10-7 M IgE S + 1.0 × 10-6 M IgE S + 1.0 × 10-6 M IgE + 1.0 × 10-6 M [Fe(CN)6]4S + 1 .0 × 10-6 M IgE + 5.0 × 10-5 M [Fe(CN)6]4-

It is well-known that a high ionic strength is disadvantageous to the binding of aptamer and protein.5 The results from our previous AFM force measurements have also shown that the binding strength of IgE to its aptamer is reduced in the presence of metal ions, and the effect of a divalent ion is stronger than that of a monovalent ion.17 For aptamer-based luminescence analysis under the same experimental conditions as those shown in Figure 4, with an increasing concentration of monovalent Na+ ion (from 40 to 1040 mM) or divalent Mg2+ ion (from 0 to 10 mM), the maximum luminescence signal change after IgE addition reduced (from a 35% decrease to a 10% decrease) and the detection limit of IgE increased obviously (from 100 pM to around 5 nM, see the Supporting Information). However, in the physiological buffer having 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2, the sensitivity of IgE (500 pM) was still good enough for its future application in the direct detection of IgE in biological samples without sample pretreatment. This is because the ionic strength of the buffer used in the original SELEX selection of the IgE aptamer is close to that of the physiological buffer.16 Signaling Mechanism. To understand how the binding of the protein to the aptamer affects the luminescence of the [Ru(phen)2(dppz)]2+, luminescence lifetime measurements were conducted. It is known that [Ru(phen)2(dppz)]2+ has two distinct binding modes with DNA, thus displaying a biexponential emission decay.12 Both modes are intercalative in nature, with the difference between the two being the extent of the exposure of the dppz nitrogen to water. The less exposed dppz structure possesses a longer lifetime and higher luminescence. The lifetime measurements for [Ru(phen)2(dppz)]2+/aptamer are summarized in Table 1. It shows that the addition of IgE did not change the emission lifetimes of [Ru(phen)2(dppz)]2+, indicating that the binding mode of the [Ru(phen)2(dppz)]2+ complex to the aptamer did not change. However, with an increase of protein, the preexponential factors of the long lifetime component increased (from 66.7% to 80.4%) while that of the short lifetime component decreased (from 33.3% to 19.6%). Considering the decrease of the luminescence intensity with IgE mentioned above, these results suggest that the number of [Ru(phen)2(dppz)]2+ complexes intercalating with the aptamer decreases, especially for the short lifetime component. The fact that the anionic quencher [Fe(CN)6]4- did not have an obvious effect on the luminescence after the aptamer/protein complex was formed confirmed that the [Ru(phen)2(dppz)]2+ probes which remained bound to the aptamer/protein were still embedded in the aptamer helix. Therefore, the decrease in the luminescence intensity change upon protein binding results from the decrease of the Ru(II) probes intercalating with the aptamer, most probably due to the IgE-induced rearrangement of the loopstem structure of the aptamer or the blocking of the intercalation access by IgE. 5234 Analytical Chemistry, Vol. 76, No. 17, September 1, 2004

short

long

τ (ns)

%

τ (ns)

%

113 ( 3.9 102 ( 4.6 100 ( 5.3 100 ( 5.5 94.8 ( 4.5

33.3 26.6 19.6 19.9 20.7

536 ( 10 504 ( 9.4 535 ( 8.9 544 ( 9.3 554 ( 8.1

66.7 73.4 80.4 80.1 79.3

Figure 6. Titration of the [Ru(phen)2(dppz)]2+/aptamer with PDGFBB in the concentration range of 0-50 nM. Inset: the linear relationship of the luminescence change of the [Ru(phen)2(dppz)]2+/ aptamer at 610 nm and the PDGF-BB concentration in range of 0-10 nM (the y-intercept and slope of the linear curve were 0.0081 ( 0.0018 and 0.0087 ( 0.00030, respectively). The physiological buffer was used. Other conditions were the same as those in Figure 2.

Detecting Other Proteins. To illustrate whether our new signaling strategy is applicable to the detection of other proteins, another 35-nt DNA aptamer for oncoprotein PDGF-BB (Figure 1C) was then tested. PDGF-BB is a cancer-related protein which has been directly implicated in the cell transformation process and in tumor growth and progression.18 [Ru(phen)2(dppz)]2+ intercalated with the PDGF-BB aptamer possessing a three-way helix structure to emit luminescence. Its luminescence intensity also decreased with PDGF-BB in a dose-dependent manner. A titration curve of the luminescence change correlated to PDGFBB concentration (0-50 nM) in the physiological buffer is shown in Figure 6. There was a good linear relationship between the luminescence signal change and the PDGF-BB concentration (with a correlation coefficient of 0.992, Figure 6 inset). The detection limit of PDGF-BB in both physiological buffer and 1% serum was 1.0 nM. This is better than our previously reported 2 nM sensitivity using the fluorescence anisotropy method under the same experimental conditions.5 It is known that most protein aptamers that have been selected so far by in vitro SELEX are not DNA but RNA molecules. We then chose an RNA aptamer against R-thrombin, a pivotal enzyme in the regulation of thrombosis and hemostasis, to further test the generalizability of our method.19 The RNA aptamer has an estimated loop-stem structure similar to that of the IgE DNA aptamer (Figure 1D). Its stem is composed of about 11 base pairs, allowing the intercalating of [Ru(phen)2(dppz)]2+ into the hydrophobic pocket to fluoresce. Figure 7 shows the decrease of

Figure 7. Titration of the [Ru(phen)2(dppz)]2+/aptamer with R-thrombin. Inset: the linear relationship of the luminescence change and the R-thrombin concentration in the subnanomolar range (the yintercept and slope of the linear curve were 0.012 ( 0.011 and 0.025 ( 0.0019, respectively). The concentrations of the aptamer and [Ru(phen)2(dppz)]2+ were 1 × 10-8 M and 1 × 10-7 M, respectively.

luminescence intensity versus the concentration of R-thrombin and the linear calibration curve for R-thrombin quantitation (with a correlation coefficient of 0.990). With this new signaling method, 10 pM R-thrombin was detected, which was higher than the most reported sensitivity when thrombin DNA aptamers were used.4,7 The above results demonstrated that the generalizability of the signaling strategy is quite encouraging. Since RNA is susceptible to degradation by the endogenous ribonucleases typically found in serum, we have not attempted to obtain a quantitation curve for thrombin in serum. It may be possible in the future to use RNA with modified nucleotides or to add ribonuclease inhibitors to a real biological sample, such as serum, for RNA aptamer-based detection. DISCUSSION It is generally believed that the conformation of an aptamer usually becomes more compact upon protein binding.6,7 However, our results show that even with a compact conformation, the presence of protein may be unfavorable to the intercalating of [Ru(phen)2(dppz)]2+ to aptamer. The change of the [Ru(phen)2(dppz)]2+ intercalation results in a sensitive luminescence change of this light switch probe. The luminescence change, although a signal reduction, is significant enough to allow highly sensitive protein detection in real time. A previous study12 has shown that one Ru(II) light switch probe binds 3-4 bp in duplex DNA, and the same dye to base pair ratio was assumed in our study. It seems that aptamers having more base pairs and higher affinity to the proteins are advantageous for the signaling as we found that IgE sensitivity was decreased when other IgE aptamers with a reduced stem or higher dissociation constant were used. However, the (20) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland, R. P.; Mathies, R. A.; Glazer, A. N. Nucleic Acids Res. 1992, 20, 2803-2812; Glazer, A. N.; Rye, H. S. Nature 1992, 359, 859-861.

detailed mechanism of the luminescence change of the [Ru(phen)2(dppz)]2+/aptamer is still under investigation. We further found that other DNA intercalating dyes, such as TOTO,20 could also be used to detect protein as the luminescence signal of the TOTO/aptamer decreased upon IgE addition (see the Supporting Information). However, the sensitivity of IgE is lower in the TOTO system. Since the luminescence mechanism of TOTO is similar to that of [Ru(phen)2(dppz)]2+, it is expected the IgE detection based on the two DNA intercalating dyes followed the same principle. A possible reason for the lower sensitivity with TOTO is that their luminescence is less sensitive to the aptamer conformation change as they bind to DNA on the strand surface as well as in the intercalation form. In addition, the stokes shift of [Ru(phen)2(dppz)]2+ (160 nm) is much larger than that of the other dyes (e.g., 20 nm for TOTO), resulting in a lower background and thus better sensitivity. CONCLUSION We have developed a novel method for protein detection based on the [Ru(phen)2(dppz)]2+ molecular light switch and aptamer. The method takes advantage of the sensitive luminescence change of [Ru(phen)2(dppz)]2+ upon aptamer-protein binding. This labelfree method is simple, yet highly sensitive and selective. It has been successfully applied to the specific detection of three diseaserelated proteins, demonstrating its potential to be a generalized method in sensitive protein assay. The method worked well in the milieu of a complex biological sample and, more importantly, was applicable to RNA aptamers which have a larger population than DNA aptamers. It can be used not only in a homogeneous assay format, but also for aptamer-based biosensors by immobilization of the aptamers onto a solid surface. This offers a new approach to generating a wide variety of signaling aptamers for in vitro or in vivo protein monitoring. ACKNOWLEDGMENT We thank Dr. Liansheng Ling and Professor Zhike He (Wuhan University, China) for providing [Ru(phen)2(dppz)]2+. This work was supported by the National Natural Science Foundation of China (Nos. 20225516, 20121301) and the Chinese Academy of Sciences. SUPPORTING INFORMATION AVAILABLE Luminescence emission spectra of [Ru(phen)2(dppz)]2+ with the dsDNA or ssDNA in the control experiments (Supporting Figure 1); luminescence emission spectra of [Ru(phen)2(dppz)]2+/ aptamer in serum (Supporting Figure 2); titration of PDGF-BB in 1% serum (Supporting Figure 3); titration of the TOTO/aptamer with IgE in the concentration range of 0-10 nM (Supporting Figure 4); the detection limits of IgE obtained under different concentrations of NaCl in the PBS buffer (Supporting Figure 5). This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 21, 2004. Accepted June 7, 2004. AC049565U

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