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Institute of Environmental Medicine, Tongji Medical College, Huazhong University of ... 13 Hangkong Road, Wuhan 430030, P. R. China, and Institute of ...
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Anal. Chem. 2004, 76, 5605-5610

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Ultrasensitive Detection of Protein Using an Aptamer-Based Exonuclease Protection Assay Xiao-Li Wang,† Fang Li,† Yan-Hua Su,† Xi Sun,† Xiao-Bo Li,† Hermann J. Schluesener,‡ Fei Tang,† and Shun-Qing Xu*,†

Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, P. R. China, and Institute of Brain Research, University of Tuebingen, Calwer Strasse 3, D-72076 Tuebingen, Germany

Currently, methods for protein detection are not as sensitive and specific as methods for detection of specific nucleic acid sequences. Here, we present an analogous technique for detection of proteins using aptamers as ligands for target binding. We have named this method the aptamer-based exonuclease protection assay. We applied a special oligonucleotide probe containing a thrombin aptamer, which has the capacity to recognize thrombin with high affinity and specificity. The aptamer probe is a 22-base-long single-strand oligonucleotide with the thrombin aptamer sequence at the 3′-terminus and 7 additional nucleotides at the 5′-terminus, which is able to bind thrombin with high affinity and specificity. In the exonuclease protection assay, thrombin binds the aptamer and thereby protects it from degradation by exonuclease I, whereas any unbound aptamer probe is degraded by exonuclease I. Subsequently, the aptamer probes that were protected from exonuclease I by thrombin act as linkers to join two free connectors, which contain sequences matching the probe. The joined products, which reflect the identity and amount of the target protein, are amplified by PCR. The exonuclease protection assay is extremely sensitive, since it is based on PCR amplification. This method can detect as few as several hundred molecules of target protein without using washes or separations. In addition, this new method for protein detection is simple and inherits all the advantages of * To whom correspondence should be addressed. Tel: 86 27 83692721. Fax: 86 27 83657705. E-mail: [email protected]. † Huazhong University of Science and Technology. ‡ University of Tuebingen. 10.1021/ac0494228 CCC: $27.50 Published on Web 08/24/2004

© 2004 American Chemical Society

aptamers. The mechanism, moreover, may be generalized and used for other forms of protein analysis. The detection and quantification of proteins play essential roles in basic discovery research as well as in clinical practice. However, methods for protein detection are not as sensitive or specific as methods for detection of specific nucleic acid sequences. Today, immunological assays, based on the use of antibodies, are the most commonly used diagnostic formats. Aptamers, though, can rival antibodies in diagnostics. Aptamers are RNA or DNA oligonucleotides originating from in vitro selection experiments (termed SELEX: systematic evolution of ligands by exponential enrichment), which start from random sequence libraries, and deliver optimized nucleic acids for high-affinity binding to given ligands such as proteins.1 Although aptamers are different from antibodies, they mimic properties of antibodies in a variety of diagnostic formats.2 The demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill the requirement of molecular recognition in those assays.3 Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. Several methods for protein detection using aptamers have been developed. German et al. applied fluorescent-labeled aptamers to affinity capillary electrophoresis.4 Drolet et al. described an enzyme-linked sandwich assay using aptamer for detection of hVEGF.5 Fang et al. developed a fluorescence anisotropy assay (1) Hermann, T.; Patel, D. J. Science 2000, 287, 820-825. (2) Jayasena, S. D. Clin. Chem. 1999, 45, 1628-1650. (3) Famulok, M.; Mayer, G.; Blind, M. Acc. Chem. Res. 2000, 33, 591-599. (4) German, I.; Buchanan, D. D.; Kennedy, R. T. Anal. Chem. 1998, 70, 45404545. (5) Drolet, D. W.; Moon-McDermott, L.; Romig, T. S. Nat. Biotechnol. 1996, 14, 1021-1025.

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for PDGF using a fluorescein-labeled single-strand DNA aptamer.6 Several molecular aptamer beacons have been designed for the detection of proteins.7-10 Fredriksson et al. developed a highly sensitive detection method for PDGF based on aptamer, termed proximity ligation assay, which can detect zeptomole (40 × 10-21mol) amounts of PDGF.11 Although the proximity ligation assay is very sensitive and detects zeptomole amounts of protein, it can only detect homodimer protein targets (e.g., platelet-derived growth factor), which are able to bind two aptamers at two distinct sites. Thus, its application range is greatly limited. To detect general protein targets, we developed an exonuclease protection assay (EPA) based on the use of aptamers as ligands. The method depends on the protection of aptamer oligonucleotides from exonuclease I degradation through binding of the aptamers with their target molecules. An aptamer-derived oligonucleotide containing a thrombin aptamer, which has the capacity to recognize thrombin with high affinity and specificity, was used as a probe in this exonuclease protection PCR assay. Thrombin binds the aptamer and protects it from degradation by exonuclease I, whereas the aptamer probes that are not bound by thrombin are degraded by exonuclease I. Subsequently, the aptamer probes protected from exonuclease I by thrombin reflect the identity and amount of target protein and can be quantified by real-time PCR. EXPERIMENTAL SECTION Material. All oligonucleotide sequences were customerdesigned and synthesized by Bioasia Biotechnology Inc (Shanghai, China). Human thrombin, BSA, and factor IX were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). Exonuclease I was from Promega (Promega Co., Madison, WI) and T4 DNA ligase from New England Biolabs (Beverly, MA). All reagents for PCR were purchased from Sino-American Biotechnology Co. (Beijing, China). The LightCycler DNA Master SYBR Green I kit was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Superpurified water was used to prepare all the solutions. The LightCycler used is from Roche Diagnostics. The results were analyzed with the LightCycler software program version 3.5 (Roche Diagnostics). Exonuclease I Protection Assay. The aptamer probe (0.1 pmol) in 10 µL of binding buffer (20 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mg/ml tRNA) was incubated with or without thrombin (1µL of 2.75 × 10-14-2.75 × 10-8 M) at room temperature for 1 h to allow complete binding. Then, 20 units of exonuclease I was added to the mixture in order to degrade the unbound probe and the resultant mixture incubated at 37 °C for 2 h, followed by a heating step at 80 °C for 15 min to inactivate the exonuclease I. After this step, two connectors (4 pmol, respectively) and T4 DNA ligase were added, and the mixture was incubated at 37 °C for 20 min and subsequently (6) Fang, X.; Cao, Z.; Beck, T.; Tan, W. Anal. Chem. 2001, 73, 5752-5757. (7) Fang, X.; Mi, Y.; Li, J. J.; Beck, T.; Schuster, S.; Tan, W. Cell. Biochem. Biophys. 2002, 37, 71-81. (8) Li. J. J.; Fang, X.; Tan, W. Biochem. Biophys. Res. Commun. 2002, 292, 31-40. (9) Hamaguchi, N.; Ellington, A.; Stanton, M. Anal. Biochem. 2001, 294, 126131. (10) Yamamoto, R.; Baba, T.; Kumar, P. K. Genes Cells. 2000, 5, 389-396. (11) Fredriksson, S.; Gullberg, M.; Jarvius, J.; Olsson, C.; Pietras, K.; Gustafsdottir, S. M.; Ostman, A.; Landegren, U. Nat. Biotechnol. 2002, 20, 473-477.

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heated to 65 °C to inactivate the T4 ligase. A 1-µL aliquot of the mixture was used for PCR amplification. The PCR program consisted of 23 cycles of denaturation (94 °C, 20 s), reannealing (62 °C, 20 s), and extension (72 °C, 30 s), using 0.5 mM concentration of each primer in a total PCR volume of 50 µL. The PCR products were analyzed by agarose gel (2.5%) electrophoresis. Fluorescent Quantitative PCR. Fluorescent quantitative PCR permits the real-time monitoring of the PCR, since the fluorescent signals are proportional to the concentration of the product, can be measured once per cycle, and are immediately displayed on a computer screen. After amplification, the temperature is slowly elevated above the melting temperature of the PCR product to measure the fluorescence for the melting curve. This enables the identification of specific DNA sequences because specific and nonspecific products have different melting temperatures, depending on their nucleotide composition. According to the protocol of the LightCycler DNA Master SYBR Green I kit, the PCR reaction mixture included the following: 2 µL of the LightCycler DNA Master SYBR Green I mix, 2.4 µL of MgCl2 (25 mM), 0.5 µL of upstream and downstream primers, respectively (10 pmol/µL), 1 µL of template, and 13.6 µL of sterile distilled water. The PCR protocol consisted of an initial denaturation at 95 °C for 2 min followed by 35 cycles at 95 °C for 0 s, 62 °C for 5 s, and 72 °C for 10 s (using a slope of 20 °C/s), to detect and quantify the fluorescence at a temperature above the denaturation temperature of primer-dimers. A melting curve temperature profile was obtained by incubating at 95 °C for 0 s and 65 °C for 15 s using a slope of 20 °C/s, followed by 95 °C for 0 s, and finally cooling at 40 °C for 30 s. Fluorescence was measured after each annealing step. Each sample was run in triplicate, and threshold cycle (CT) values were averaged from all reactions. RESULTS AND DISCCUSSION Design Strategy of Exonuclease Protection Assay. Aptamers are highly selective for their targeted ligands and have greater binding constants than the original oligonucleotide mixture.12 Exonuclease I catalyzes the removal of nucleotides from singlestranded DNA in the 3′ to 5′ direction. Without a protein target, the aptamer probe would be degraded to 5′-terminal dinucleotides with the release of deoxyribonucleoside 5′-monophosphates from the 3′-termini of the single-stranded DNA chains. In the assay described here, the oligonucleotides that bind thrombin are protected, whereas free single-strands are completely degraded by exonuclease I. Then the two connectors are joined by the protected probes to form longer oligonucleotide strands for PCR reaction. Last, the ligated products are quantified by real-time PCR. Therefore, the amount of ligated products corresponds to the amount of the protected probes and indirectly reflects the amount of thrombin. (Figure 1). In this study, a thrombin aptamer was used as affinity probe to detect human thrombin, a critical enzyme in the blood coagulation system. The thrombin aptamer is a well-known selective product of SELEX with a binding affinity Kd of 10-150 nM.9,13 The core sequence of this aptamer is GGT TGG TGT GGT (12) Green, L. S.; Jellinek, D.; Jenison, R.; Ostman, A.; Heldin, C. H.; Janjic, N. Biochemistry 1996, 35, 14413-14424. (13) Bock, L. C.; Griffin, L. C.; Latham, J. A.; Vermaas, E. H.; Toole, J. J. Nature 1992, 355, 564-566.

Figure 1. Detection flowchart of the exonuclease protection assay. (1) Thrombin aptamers adequately bind thrombin in the proper buffer. (2) Exonuclease I is added to hydrolyze free aptamers. (3) Two additional oligonucleotide strands, named connectors, are subsequently put into the reaction mix and allowed to hybridize with the aptamers. (4) T4 DNA ligase is used to ligate the hybridization products. (5) The ligation products, which reflect the identity and amount of protein, are quantified through fluorescent quantitative PCR..

TGG. In a proper solution, such as physiological buffer, it folds into a chairlike structure with two G-tetrad stacks by two TT loops and a single TGT loop,14,15,16 which plays an important role in binding to thrombin. In this experiment, exonuclease I was used to degrade free aptamer probe. The time of exonuclease digestion is pivotal to quantify analyte protein because exonuclease I will drive the equilibrium between the bound and unbound aptamer toward the unbound aptamer. Although the decrease of free aptamer is fast at first, the drive of this equilibrium is slow because of such a small Kd between 10 and 150 nmol/L; then the concentration of free aptamers was so low with the reference of the negative control that the reaction velocity of exonuclease I was extremely slow. Within limited time, there is very little effect of exonuclease on the moving of this equilibrium. After the same time of digestion, the concentrations of free aptamer without binding thrombin in each sample are extremely low and almost the same. The proportions of bound thrombin to total thrombin in each sample are almostly unvaried during the limited time. The sequences of the aptamer probe and the two connectors used in this study are 5′GGT TGGT GGT TG G TGT GGT TGG3′ (aptamer probe, the underline is the aptamer sequence), 5′CAG CCA ACA GGC ACC GAA CCA ACC ACC AAC 3′(upstream connector, the sequence in italics matches the one in italics of the probe) and 5′p-C ACA CCA ACC TTC GGC ACC AAG CTA GTC GAT 3′ (downstream connector, the boldface portion matches the boldface sequence of the probe), respectively. It is important that, during the design of the oligonucleotides, stem-loop structures due to self-hybridization between the oligonucleotide fragments should be avoided, because exonuclease I cannot degrade double-strand DNA.17 All the single DNA strands above were checked for the absence of secondary structure formation on themselves or between each other (certain (14) Griffin, L. C.; Toole, J. J.; Leung, L. L. Gene 1993, 137, 25-31. (15) Wang, K. Y.; McCurdy, S.; Shea, R. G.; Swaminathan, S.; Bolton, P. H. Biochemistry 1993, 32, 1899-1904. (16) Wang, K. Y.; Krawczyk, S. H.; Bischofberger, N.; Swaminathan, S.; Bolton, P. H. Biochemistry 1993, 32, 11285-11292. (17) Kushner, S. R.; Nagaishi, H.; Templin, A.; Clark, A. J. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 824-827.

software on the web of www.bioinfo.rpi.edu). The aptamer probe was designed to include one thrombin aptamer and 7 extra oligonucleotides. These additional oligonucleotides favor the hybridization with the connectors; however, a higher number of additional oligonucleotides would enhance the risk of nonspecific hybridization. For this assay, the standard curve was based on the analysis of external standards. Since the accuracy of quantification with external standards depends on the standards, we synthesized, to minimize variation, an oligonucleotide with the same sequence of the thrombin ligations as the standard template. Detection of Thrombin by EPA-PCR. Serial dilutions of the standard were amplified by conventional PCR. Simultaneously, ligation products obtained with the exonuclease protection assay on various amounts of thrombin were also amplified by PCR. All the products were analyzed by gel electrophoresis (See Figure 2.). As shown in Figure 2, the intensity of the corresponding bands increased with the increase of concentrations of either standards or thrombin for the lower concentrations. However, when the concentration reached a certain level, there was no difference between the bands; this indicates that most likely the PCR reaction reached its maximum. Therefore, we could only detect qualitative differences in the amount of thrombin by gel analysis. Quantification of Thrombin by EPA Real-Time PCR. The 100-fold serial dilutions, in the range of 10-15-10-9M (6 × 102-6 × 108 copies/µL), of a template with the same sequence as the ligation product were used to generate a standard curve for realtime PCR (See Figure 3.). The CT values (y-axis) were plotted against the corresponding logarithm of the template concentration (x-axis). CT values, namely, threshold cycle values, were defined as the cycle at which SYBR Green I fluorescence increased significantly above the background as a result of amplicon formation. The line was described using the equation y ) kx + b, where y is the value of CT, x is the logarithm of the template copy number, k is the slope, and b is the y-intercept. In this study, the equation was y ) -2.95x + 33.65 with R2 ) 0.990, which showed a good correlation between the amount of the starting copies of the standard and the CT values. Then we further diluted the amount of the standard templates to amplify and found that there Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

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Figure 2. Detection of PCR products of serial dilutions of the standard or of ligation products obtained in the protection assay by 2.5% agarose gel electrophoresis. (A) PCR products of the standard. Lanes; 1, 25-bp ladder; 2, negative control; 3, 10-9 M; 4, 10-11 M; 5, 10-13 M; 6, 10-15 M. (B) PCR products after amplification of ligation products obtained in the thrombin assay. Lanes: 1, 25-bp ladder; 2, negative control; 3, thrombin 1:106 diluted; 4, thrombin 1:104 diluted; 5, thrombin 1:102 diluted; 6, thrombin undiluted.

Figure 3. Standard curve obtained by ploting the copies of the standard versus the threshold cycle CT. Synthesized DNA templates means the standard and samples were synthesized by the incorporation.

was a good linear relationship at the range from 6 × 102 to 6 × 108 copies. We set up four dosage groups each containing 1 µL of thrombin with a concentration range of 2.75 × 10-14-2.75 × 10-8 M with steps of 2 orders of magnitude. Ligation products obtained from serial thrombin dilutions were detected by Q-PCR. The results included the amplification curve and melting curve (Figure 4 and Figure 7B). By comparing the CT of the various groups with the standard curve, we deduced the amount of ligation product for every group, which indirectly illustrated the counterpart amount of thrombin. Then we constructed a standard curve with the amount of thrombin and the corresponding ligation product (Figure 5). The resulting equation was y ) 2.0653x0.826 with correlation coefficient R2 ) 0.998. This equation shows the exponential relationship between ligation products and thrombin with good accuracy especially at low concentration. The predictive value of thrombin by exponential plot is more accurate than that by linear plot especially from 5.4 × 102 to 5.4 × 105 molecules of thrombin. Ranging from 5.4 × 102 to 5.4 × 108 starting molecules of thrombin, the assay showed a reliable and sensitive quantification of thrombin with a good fit (R2 ) 0.998). In this assay, fixing the aptamer probe amount for about 0.1 pmol and 2-fold the CT value of the background as the criterion, we can detect as few as several hundred molecules for thrombin by EPA. Compared with the previously reported method for thrombin detection with the 3 orders of magnitude and 0.7 amol (10-18) of molecules detection limit by aptamer biosensors,18 the EPA improves greatly in detection range and detection limit. (18) Potyrailo, R. A.; Conrad, R. C.; Ellington, A. D.; Hieftje, G. M. Anal. Chem. 1998, 70, 3419-3425.

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Figure 4. Amplification curves observed by real-time PCR using SYBR Green I. Templates were ligation products obtained after thrombin protection. 100-fold serial dilutions of thrombin were used. The negative control consisted of sterile water.

Figure 5. Thrombin detection standard curve obtained by exonuclease protection assay. The logarithm of the ligation products was plotted against the logarithm of the known quantity of thrombin.

Because the Q-PCR is highly sensitive, it is critical to cautiously execute the experiments to avoid contamination among samples. Furthermore, random error variation due to sampling errors also becomes important. All the samples in the assay were analyzed three times with the variation coefficient less than 5%, which showed high repeatability of this method. Optimization of the EPA. In this assay, the aptamers that are not bound to thrombin must be completely degraded or there

Figure 6. Optimization of the reaction conditions for the EPA: analysis of the PCR products by agarose gel (2.5%) electrophoresis. (A) Effect of the ratio of aptamer probe concentration to connector oligonucleotide concentration (part of results shown in the picture). Lanes: 1, 1:10 negative; 2, 1:10, positive group; 3, 1:60 positive group; 4, 1:60 negative control; 5, 1:40 positive group; 6, 1:40 negative group. (B) Effect of exonuclease I action time on the negative control in a fixed reaction mixture (0.1 pmol of aptamer probe, 4 pmol of connectors in total volume 30 µL). Lanes: 1, 0.5 h; 2, 1 h; 3, 1.5 h; 4, 2 h; 5, 12 h. (C) Effect of the ligation time on the assay. The reaction mixture included 0.1 pmol of aptamer probe, 4 pmol of connectors, and 1 µL of 2.75 × 10-14 M thrombin (positive group) or 1 µL of sterile diluted water (negative control) in a total volume of 30 µL. Lanes: 1, 30 min positive; 2, 30 min negative; 3, 10 min positive; 4, 10 min negative; 5, 20 min positive; 6, 20 min negative.

will be a high background, which will affect the precision. So it is important to determine the optimal reaction mixture and reaction time in order to reduce the background interference. First, a fixed amount of thrombin was incubated with various amounts of aptamer and connectors for a set reaction time. To ensure efficient formation of ligation substrates, the connectors should be in large molar excess over the aptamer probe. An increase in the proportion of aptamer probes to connectors resulted in increasingly brighter bands in agarose gel analysis (Figure 6A). At ratios of more than 1:40 of connectors to probe, there was no difference in the brightness of the bands anymore; however, there was a higher background. Therefore, the ratio of 1:40 was chosen as the optimal reaction parameter for the molar ratio of probe to connectors. On the other hand, a high concentration of free aptamer probes might be degraded incompletely, which could lead to an overestimation of the sample amount. So on the premise that thrombin completely binds the aptamer probe, the amount of aptamer probe should be as small as possible. Subsequently, the amounts of thrombin and all reagents in the experiment were kept constant whereas the reaction times of exonuclease I or DNA ligase treatment were varied (Figure 6B,C). We found that, the longer the incubation with exonuclease I, the more free aptamer probes were degraded. In the negative control, a band corresponding to the target strands was visible at reaction times of exonuclease I shorter than 2 h; therefore free aptamers were not degraded completely in less than 2 h. Thus, 2 h is the optimal reaction time for exonuclease I. We also found that both the signal and background increased with the extension of DNA ligation. A total of 20 min gave the optimal signal/background noise ratio in all reactions and was therefore chosen as the reaction time for DNA ligation in this assay. Specificity of the Aptamer Probe. To estimate the specificity of the assay, we used BSA and factor IX as controls. Factor IX is plasma serine protease and is ∼37% identical to thrombin in its catalytic domain.9 BSA and IX were incubated individually with the thrombin aptamer probe and processed in the same way as thrombin. The PCR reactions were checked by both gel analysis and melting curve (See Figure 7.). No bands were observed after gel electrophoresis, and melting temperatures were different from the one for thrombin. Thus, no significant cross-reactivity was detected for these proteins, which demonstrated that the aptamer probe exhibited a high degree of selectivity for thrombin.

Figure 7. Specificity analysis of the exonuclease protection assay. (A) Agarose gel (2.5%) electrophoresis of PCR-amplified products of different protein ligation products. Lanes: 1, 25-bp ladder; 2, negative control; 3, thrombin; 4, BSA; 5, factor IX. There were no bands with BSA or factor IX, while there was the expected band in the thrombin sample, which showed that BSA and factor IX do not lead to the same ligation products as thrombin. (B) Melting curves from real-time amplification of protein ligation products using SYBR green I: 1, negative control; 2, BSA; 3, factor IX; 4, thrombin 1:106 diluted; 5, thrombin 1:104 diluted; 6, thrombin 1:102 diluted; 7, thrombin undiluted. The melting curves of BSA and IX were different from those of thrombin, which suggests that no nonspecific reactions were observed with this method.

Application of the EPA. Taking advantage of the special nature of aptamers and exonuclease I, we developed a protein detection method, the exonuclease protection assay. Compared with other methods for protein detection, the probes and proteins in EPA need no special disposal such as labeling,7-10 immobilization,18 and coating,19 which greatly decreases the operating difficulty. Moreover, the assay detects proteins without steps increasing the loss of the protein such as washing or separation. It only consists of a relatively short preincubation period of the samples with the aptamer probes, followed by the addition of the reagents required for degradation and ligation and subsequent real-time detection by fluorometric quantitative PCR. (19) Jiang, Y.; Zhu, C.; Ling, L.; Wan, L.; Fang, X.; Bai, C. Anal. Chem. 2003, 75, 2112-2116.

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The special commercial instrument prerequisite, LightCycler, will be available in most laboratories with the improvement of quantitative techniques. So the assay is one of fast determination, convenient performance, and low cost. The detection range of the assay can reach 7 orders of magnitude, and the number of thrombin molecules that could be detected was on the order of 102. Compared with traditional detection methods of protein such as ELISA,20 both the sensitivity and the linear range advance enormously. Although having a sensitivity similar to the recently developed proximity ligation method,11 which requires simultaneous and proximate recognition by pairs of affinity probes, our method does not require such restriction of aptamer and probably applies to more aptamers. Therefore, it might have a wider application range than the proximity ligation assay. Up to now, ∼100 aptamers with good affinity and specificity for proteins have been reported in the literature.21 So it has a promising future in the field of proteomics research. On the other hand, several limitations also exist. The serious shortcomings lie in the incomplete degradation of the high concentration of free aptamer probes, which could lead to an overestimation of the sample amount. The discovery of new nucleases or cooperation with more nucleases may be an excellent way to increase the degradation efficiency. Furthermore, there (20) Wieding, J. U.; Hosius, C. Thromb. Res. 1992, 65, 745-756. (21) Brody, E. N.; Willis, M. C.; Smith, J. D.; Jayasena, S.; Zichi, D.; Gold, L. Mol. Diagn. 1999, 4, 381-388.

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are differences in experimental conditions such as reagent concentrations and reaction time for different aptamers. So it is necessary to optimize the reaction conditions for each specific protein. CONCLUSION An aptamer with the ability to interact with nucleic acids as well as other classes of targets was used to close the communication gap between oligonucleotides and proteins. A new strategy for protein detection based on aptamers has been described here. This method is highly sensitive, specific, and widely available, shows a good linearity and repeatability, and allows relative accurate quantification down to a few molecules. This protein assay, based on protection of the aptamer by its target protein, not only serves as ultrasensitive detection of biological molecules but also offers outstanding prospects for the determination of protein-nucleic acid interactions, binding stoichiometry, and DNA damage. ACKNOWLEDGMENT We thank Dr. Yi-Rong Li, Union Hospitial, Wuhan, for his help in real-time PCR. This work was supported by National Natural Science Fundation of China (30170051 and 30170903). Received for review April 16, 2004. Accepted July 11, 2004. AC0494228