- Email: [email protected]
Molecular beacon-based quantitiation of epithelial tumor marker mucin 1
Bioorganic & Medicinal Chemistry Letters 22 (2012) 6081–6084
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Molecular beacon-based quantitiation of epithelial tumor marker mucin 1 Seonmi Shin a, , Hye Yeon Nam a, , Eun Jeong Lee a, , Woong Jung b, Sang Soo Hah a,⇑ a b
Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, South Korea Department of Emergency Medicine, Kyung Hee University Hospital at Gangdong, Seoul 134-727, South Korea
a r t i c l e
i n f o
Article history: Received 9 July 2012 Revised 7 August 2012 Accepted 10 August 2012 Available online 16 August 2012 Keywords: Molecular beacon FRET Protein quantitation Mucin 1
a b s t r a c t Mucin 1 (Muc1) is a glycoprotein expressed on most epithelial cell surfaces, which has been confirmed as a useful biomarker for the diagnosis of early cancers. In this study, we demonstrate that a quantum dot (QD)-aptamer beacon acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3, in the presence of the target molecules, Muc1. Release of intercalated BOBO-3s from the QD-conjugated aptamers results in a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission, allowing for label-free Muc1 detection and quantitation. We attain highly specific and wide-range detection (from 50 nM to 20 lM) of Muc1, suggesting that our QD-aptamer beacon can be a potential alternative to immuno-based assays for Muc1 detection. The detection methodology is expected to be improved for the early diagnosis of different types of epithelial cancers of large populations. Ó 2012 Elsevier Ltd. All rights reserved.
Mucins, cell-surface associated glycoproteins, are bound to cells by an integral transmembrane domain.1 Among the family, mucin 1 (Muc1) contains a hydrophobic membrane-spanning domain of 31 amino acids, a cytoplasmic domain of 69 amino acids, and an extracellular domain consisting of a region of nearly identical repeats of 20 amino acids per repeat.1 Muc1 has a protection function as its biological role by binding to pathogens and possibly functions in a signal transduction pathway.2 Muc1 is overexpressed in almost all human epithelial cell adenocarcinomas, including breast,3 gastric,4 colorectal,5 lung,6 prostate,7 ovarian,8 pancreatic,9 and bladder carcinomas.10 Moreover, the expression of Muc1 in these tissues usually lacks regular expression patterns, making the protein ubiquitously and randomly expressed all over the cell surface.11 Thus, large amounts of the protein can be found in blood of cancer patients,12 which allows serum assays for Muc1 to be potentially useful for early cancer detection. In this regard, several techniques including ELISA, dot blotting, Western blotting, immunohistochemistry and immunofluorescence have been developed for Muc1 detection and quantitation.13 In particular, two immune-based assays using anti-Muc1 antibodies have been recently developed to successfully monitor the concentration of Muc1 in serum.13 However, the production of a specific antibody for these antibody-based assays is time-consuming and not a fool proof procedure. In addition, the procedures usually use radioactively or fluorescently labeled secondary antibodies for sensitivity purposes. In the present study, we explore an aptamer-based detection methodology for Muc1 quantitation, with an ultimate goal of ⇑ Corresponding author. Tel.: +82 2 961 2186; fax: +82 2 966 3701.
E-mail address: [email protected] (S.S. Hah). These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.08.037
developing practical and quantitative analytical tools for tumor markers. Aptamers are a special class of nucleic acids that can specifically bind, with high affinity, to a wide array of target molecule.14 They have originated from large random-sequence nucleic acid libraries via an in vitro evolution process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment).15 To date, a wide range of DNA- and RNA-based aptamers have been discovered that bind with the dissociation constants ranging from nanomolar to picomolar level to metal ions,16 small molecules,17 proteins,14 and even entire cells.18 Importantly, aptamers rival antibodies, the traditional recognition molecule, because of a number of unique features of aptamers.14,19 First, the limit of having to use cell lines or animals, as is necessary for antibodies, can be overcome. Aptamers, once selected, can undergo subsequent amplification through polymerase chain reaction or transcription to produce a large quantity with high purity. Second, the simple chemical structure of aptamer makes it amendable to further modifications with functional groups based on the experimental purposes.7 Finally, aptamers are more stable than antibodies, allowing them to be suited for applications requiring harsh conditions (e.g., high temperature or extreme pH). Among a variety of aptamer-based alternatives to traditional immunoassays, molecular beacon assays are ideal for both in vitro and in vivo real-time and label-free protein sensing, because the beacon assay relies on an aptamer probe with a labeled fluorescent donor at one end and an acceptor, either a fluorophore or a quencher, at the other end,20 allowing fluorescence resonance energy transfer (FRET) between the donor and the acceptor to occur in the absence of the target. In the presence of the target, however, a binding-induced conformational change of the aptamer probe is initiated and alters the beacon’s donor– acceptor distance, leading to a change of FRET efficiency.21 Then
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S. Shin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6081–6084
Scheme 1. Label-free detection of Mucin 1 (Muc1) with beacons of QD-aptamer nanoconjugates loaded with BOBO-3s. Muc1 binding to the aptamer probes on QDs causes conformational change, resulting in dissociation of the pre-stained BOBO-3s from the QD-aptamer conjugates. Thus, Muc1 can be quantified by the increase QD emission at 565 nm and the decrease of FRET-mediated BOBO-3 emission at 605 nm.
Figure 1. (a) Fluorescence spectra of QD-aptamer nanoconjugates. (b) Fluorescence spectra of QD-aptamer nanoconjugates loaded with BOBO-3s. The intercalated dyes induce FRET-mediated decrease of QD fluorescence at 565 nm and increase of BOBO-3 fluorescence at 605 nm, which can be alternatively shown in (c) resulting from subtraction of the fluorescence intensities of QD-aptamer nanoconjugates loaded with the intercalating dyes from those of QD-aptamer nanoconjugates.
the target binding can be quantified based on the emission change at either donor’s or acceptor’s characteristic wavelength or both. An aptamer beacon, thus, has not only fluorescence sensitivity but also multi-dimensional specificity for label-free protein detection.
Whereas aptamers are used as a recognition element of the beacons, quantum dots (QDs) are used as a signal transducing element or the beacon’s FRET donor in the present study. QDs are semiconducting nanoparticles, which can be prepared with interesting optical properties.22 The fluorescent properties of QDs including phostostability, narrow emission bands, broad excitation spectra, and accessibility to versatile functionalization are the key advantages for their use as FRET donors,23 since QDs absorb light over a broad spectral range and fluoresce at wavelengths determined by their physical sizes, producing precise and narrow spectral emissions, and QDs are extremely efficient at absorbing light and converting it to a highly stable fluorescent emission.22 These niches have paved a new way to the development of QD-aptamer beacons.24 As the beacon’s FRET acceptors or reporters, we make use of a dimeric cyanine DNA intercalating dye BOBO-3 (1,10 -(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methylidene]-pyridinium tetraiodide, (excitation at 570 nm, emission at 605 nm),25 instead of additional reporter-labeled oligonucleotides to work with the aptamer probe. Our strategy is illustrated in Scheme 1. A single-stranded antiMuc1 aptamer probe is covalently conjugated to QDs (emission maxima at 565 nm).26 Then BOBO-3 that shows large fluorescence enhancement when intercalated into a double helix,27 is used to stain the duplex regions, leading to formation of QD-aptamer conjugates loaded with BOBO-3s, as clearly demonstrated in Figure 1. In the absence of the target protein, Muc1, a FRET-mediated emission of the intercalated BOBO-3s can be observed when QD is illuminated at 365 nm. Figure 1(b) shows that 365-nm excitation of the QD-aptamer nanoconjugates loaded with the intercalating dyes results in the decrease of QD fluorescence and the increase of BOBO-3 fluorescence, and that the energy transfer efficiency is estimated to be as high as 40%. Such a moderate energy transfer efficiency is attributed presumably to the fact that the thickness of the aptamer coating for dye staining (ca. 2.5 nm) is approximately a half of the Förster radius (57.7 Å) for the FRET pair of QDs and BOBO-3s.25 On the other hand, the presence of Muc1 which can bind to the aptamer, induces conformational change of the aptamer, resulting in dissociation of BOBO-3s from QD-aptamer conjugates. Thus, a decrease in the FRET-mediated BOBO-3 emission and an increase in the QD fluorescence are observed, which can allow for quantitation of the Muc1 concentration. Label-free quantitation of Muc1 in a clear buffer using our QDaptamer conjugates loaded with BOBO-3s is demonstrated in Figure 2. We measured the ratios of the emission intensities at 565 nm and 605 nm, respectively, and Figure 2(a) shows that the beacon in a buffer produces a 10–60% FRET signal change in response to 50 nM to 20 lM Muc1. It corresponds to a dynamic range across three orders of magnitude. The linear range is outlined in Figure 2(b), which demonstrates the achievement of 50 nM LOD (limit of detection).
S. Shin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6081–6084
Figure 2. Label-free quantitation of Muc1 using our QD-aptamer nanoconjugates loaded with BOBO-3s. (a) Normalized ratios of the emission intensities of QDs at 565 nm as a function of Muc1 concentration, showing that the beacon in a buffer produces a 10–60% FRET signal change in response to 50 nM to 20 lM Muc1. The data from the experiment reveal that apparent dissociation constant (Kdapp) between the QD-conjugated aptamer and Muc1 is approximately 78 pM. (b) Logarithmic scale graph for the obtained data.
To summarize, a new QD-aptamer beacon, which is advantageous over the previously reported Muc1 sensing assays in the aspect of simplicity to generate the fluorescence change from QDaptamer conjugates in a target specific manner, has been developed by using a DNA intercalating dye, BOBO-3, as a FRET reporter. The beacon is prepared by pre-staining QD-aptamer nanoconjugates loaded with BOBO-3s and acts by Muc1 binding-induced dye displacement, which shows an increase in QD emission at 565 nm and a decrease in FRET-mediated dye emission at 605 nm. Accordingly, specific and label-free Muc1 sensing with a 50 nM to 20 lM dynamic range is achieved. Our results suggest that the QD-aptamer beacon can be a potential alternative to immuno-based assays for Muc1 detection, by bringing the power of QD fluorescence technology to a work-horse application in diagnostics. It should also be noted that because of the advantageous optical properties of QDs over conventional organic dyes and a wide versatility of aptamers for selection, our detection methodology can be applied in principle to other protein biomarkers, leading to improvement for the early diagnosis of different types of epithelial cancers of large populations. Acknowledgments This work was supported by Basic Science Program through the National Research Foundation of Korea (KRF) funded by the Ministry of Education, Science and Technology (MEST) (Nos. 20110021956 and 2012-001680). References and notes 1. (a) Gendler, S. J.; Spicer, A. P. Annu. Rev. Physiol. 1997, 57, 607; (b) Hollingsworth, M. A.; Swanson, B. J. Nat. Rev. Cancer 2004, 4, 45.
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2. Wang, H.; Lillehoj, E. P.; Kim, K. C. Biochem. Biophys. Res. Commun. 2004, 321, 448. 3. (a) Hayes, D. F.; Mesa-Tejada, R.; Papsidero, L. D.; Crogham, G. A.; Korzun, A. H.; Norton, L.; Wood, W.; Strauchen, J. A.; Grimes, M.; Weiss, R. B. J. Clin. Oncol. 1991, 9, 1113; (b) Perey, L.; Hayes, D. F.; Maimonis, P.; Abe, M.; O’Hara, C.; Kufe, D. W. Cancer Res. 1992, 52, 2563. 4. Medina, M.; Velez, D.; Asenjo, J. A.; Egea, G.; Real, F. X.; Gil, J.; Subiza, J. L. Cancer Res. 1999, 1061, 59. 5. Tanimoto, T.; Tanaka, S.; Haruma, K.; Yoshihara, M.; Sumii, K.; Kajiyama, G.; Shimamoto, F.; Kohno, N. Oncology 1999, 56, 223. 6. (a) Willsher, P. C.; Xing, P.-X.; Clarke, C. P.; Ho, D. W. M.; McKenzie, I. F. C. Nat. Rev. Cancer 1993, 72, 2936; (b) Seregni, E.; Botti, C.; Lombardo, C.; Cantoni, A.; Bogni, A.; Cataldo, I.; Bombardieri, E. Anticancer Res. 1996, 16, 2209; (c) Maeshima, A.; Miyagi, A.; Hirai, T.; Nakajima, T. Pathol. Int. 1997, 47, 454. 7. Zhang, S.; Zhang, H. S.; Reuter, V. E.; Slovin, S. F.; Scher, H. I.; Livington, P. O. Clin. Cancer Res. 1998, 4, 295. 8. (a) Nacht, M.; Ferguson, A. T.; Zhang, W.; Petroziello, J. M.; Cook, B. P.; Gao, Y. H.; Maguire, S.; Riley, D.; Coppola, G.; Landes, G. M.; Madden, S. L.; Sukumar, S. Cancer Res. 1999, 59, 5464; (b) Hough, C. D.; Sherman-Baust, C. A.; Pizer, E. S.; Montz, F. J.; Im, D. D.; Rosenshein, N. B.; Cho, K. R.; Riggins, G. J.; Morin, P. J. Cancer Res. 2000, 60, 6281. 9. (a) Burdick, M. D.; Harris, A.; Reid, C. J.; Iwamura, T.; Hollingsworth, M. A. J. Biol. Chem. 1997, 272, 24198; (b) Masaki, Y.; Oka, M.; Ogura, Y.; Ueno, T.; Nishihara, K.; Tangoku, A.; Takahashi, M.; Yamamoto, M.; Irimura, T. Hepatogastroenterology 1999, 46, 2240. 10. (a) Walsh, M. D.; Hohn, B. G.; Thong, W.; Devine, P. L.; Gardiner, R. A.; Samaratunga, M. L.; McGuckin, M. A. Br. J. Urol. 1994, 73, 256; (b) Retz, M.; Lehmann, J.; Röder, C.; Plötz, B.; Harder, J.; Eggers, J.; Pauluschke, J.; Kalthoff, H.; Stöckle, M. Cancer Res. 1998, 58, 5662. 11. Baratt-Boyes, S. M. Cancer Immunol. Immunother. 1996, 43, 142. 12. (a) Beatty, P.; Hanisch, F.-G.; Stolz, D. B.; Finn, O. J.; Ciborowski, P. Clin. Cancer Res. 2001, 7, 781s; (b) Croce, M. V.; Isla-Larrain, M. T.; Demichelis, S. O.; Gori, J. R.; Price, M. R.; Segal-Eiras, A. Breast Cancer Res. Treat. 2003, 81, 195–207. 13. (a) Harrop, C. A.; Thornton, D. J.; McGuckin, M. A. Methods Mol. Biol. 2012, 842, 49–66; (b) Bon, G. G.; von Mensdorff-Pouilly, S.; Kenemans, P.; van Kamp, G. J.; Verstraeten, R. A.; Hilgers, J.; Meijer, S.; Vermorken, J. B. Clin. Chem. 1997, 43, 585–593; (c) Jezersek, B.; Cervek, J.; Rudolf, Z.; Novakovic, S. Cancer Lett. 1996, 110, 137–144; (d) Price, M. R.; Hudecz, F.; O’Sullivan, C.; Baldwin, R. W.; Edwards, P. M.; Tendler, S. J. B. Mol. Immunol. 1990, 27, 795–802; (e) Reddish, M. A.; Helbrecht, N.; Almeida, A. F.; Madiyalakan, R.; Suresh, M. R.; Longenecker, B. M. J. Tumor Marker Oncol. 1992, 7, 19–27. 14. (a) Shin, S.; Kim, I.-H.; Kang, W.; Yang, J. K.; Hah, S. S. Bioorg. Med. Chem. Lett. 2010, 20, 3322; (b) Ferreira, C. S. M.; Matthews, C. S.; Missailidis, S. Tumor Biol. 2006, 27, 289; (c) Lee, G.-H.; Hah, S. S. Bioorg. Med. Chem. Lett. 2011, 22, 1520; (d) Nimjee, S. M.; Rusconi, C. P.; Sullenger, B. A. Annu. Rev. Med. 2005, 56, 555; (e) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768. 15. Tuerk, C.; Gold, L. Science 1990, 249, 505. 16. (a) Kim, J.; Kim, M. Y.; Kim, H. S.; Hah, S. S. Bioorg. Med. Chem. Lett. 2011, 21, 4020; (b) Wang, L.; Liu, X.; Hu, X.; Song, S.; Fan, C. Chem. Commun. 2006, 3780; (c) He, F.; Tang, Y.; Wang, S.; Li, Y.; Zhu, D. J. Am. Chem. Soc. 2005, 127, 12343; (d) Ueyama, H.; Takagi, M.; Takenaka, S. J. Am. Chem. Soc. 2002, 124, 14286. 17. (a) Sankaran, N. B.; Nishizawa, S.; Seino, T.; Yoshimoto, K.; Teramae, N. Angew. Chem., Int. Ed. 2006, 45, 1563; (b) Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2006, 45, 90. 18. Fang, X.; Tan, W. Acc. Chem. Res. 2010, 43, 48. 19. Iliuk, A. B.; Hu, L.; Tao, W. A. Anal. Chem. 2011, 83, 4440. 20. Tan, W.; Wang, K.; Drake, T. J. Curr. Opin. Chem. Biol. 2004, 8, 547. 21. (a) Wang, X.; Dong, P.; Yun, W.; Xu, Y.; He, P.; Fang, Y. Biosens. Bioelectron. 2009, 24, 3288; (b) Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Angew. Chem., Int. Ed. 2005, 44, 5456; (c) Li, J.; Fang, X.; Tan, W. Biochem. Biophys. Res. Commun. 2002, 292, 31; (d) Hamaguchi, N.; Ellington, A.; Stanton, M. Anal. Biochem. 2001, 294, 126; (e) Shin, S.; Kwon, H.-M.; Yoon, K.-S.; Kim, D.-E.; Hah, S. S. Mol. Biosyst. 2011, 7, 2110. 22. (a) Alivisatos, A. P.; Gu, W.; Larabell, C. Annu. Rev. Biomed. Eng. 2005, 7, 55; (b) Alivisatos, P. Nat. Biotechnol. 2004, 22, 47. 23. (a) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538; (b) Kim, Y. S.; Kim, M. Y.; Song, J. K.; Kim, T. J.; Kim, Y. D.; Hah, S. S. Chem. Commun. 2012, 48, 723; (c) Kim, M. Y.; Kim, Y. S.; Kim, J.; Hah, S. S.; Kim, T. J.; Kim, Y. D. Biotechnol. Lett. 2011, 33, 623. 24. (a) Zhang, C. Y.; Johnson, L. W. Anal. Chem. 2009, 81, 3051; (b) Kim, G. I.; Kim, K. W.; Oh, M. K.; Sung, Y. M. Nanotechnology 2009, 20, 175503; (c) Swain, M. D.; Octain, J.; Benson, D. E. Bioconjugate Chem. 2008, 19, 2520; (d) Levy, M.; Cater, S. F.; Ellington, A. D. Chembiochem 2005, 6, 2163–2166. 25. Lim, T. C.; Bailey, V. J.; Ho, Y. P.; Wang, T. H. Nanotechnology 2008, 19, 075701. 26. Reagents were obtained from commercial suppliers and were used without further purification, and depc-treated deionized water was used for all experiments. All experiments were performed in duplicate. The QD-aptamer nanoconjugates were prepared by adapting literature procedures (Ref. 14a). In brief, QDs with emission maxima of 565 nm and modified with PEG and amino groups were obtained from Invitrogen (Carlsbad, CA). QD concentrations were measured by optical absorbance, using extinction coefficients provided by the supplier. Sulfosuccinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, Sigma) was used 0 as cross-linker. HPLC-purified RNA-based probes (5 -HS-(CH2)6-GCGAG CGCAG
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TTGAT CCTTT GGATA CCCTG GGCTC GC-30 , Ref. 14b) for specific binding to Muc1 proteins were purchased from Bioneer (Daejeon, Korea), which was used for conjugation with the activated QDs following dithiothreitol treatment. The resulting QD-aptamer products were dissolved in PBS (200 lL) and stored at 4 °C for further experiments. Prior to detection of Muc1 (Peptron, Daejeon, Korea), each QD-aptamer (with 1 lM QD) nanoconjugate was pre-treated for annealing and mixed with 10 lM BOBO-3 (Invitrogen) in PBS in the dark for 1 h. The resulting QD-aptamer conjugates loaded with BOBO-3s were separated by centrifugation at 200g
for 1 min from the unbound BOBO-3s in the supernatant. Then, a given Muc1 sample dissolved in PBS was added with a final volume of 200 lL to the QDaptamer conjugates loaded with BOBO-3s. After 1-h incubation, the mixture was transferred to a fluorescence microplate reader, to measure its FRETmediated BOBO-3 emission signal (365-nm excitation, followed by 565- and 605-nm emission). 27. Ruedas-Rama, M. J.; Orte, A.; Crovetto, L.; Talavera, E. M.; Alvarez-Pez, J. M. J. Phys. Chem. B 2010, 1094, 114.
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Molecular beacon-based quantitiation of epithelial tumor marker mucin 1 Seonmi Shin a, , Hye Yeon Nam a, , Eun Jeong Lee a, , Woong Jung b, Sang Soo Hah a,⇑ a b
Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, South Korea Department of Emergency Medicine, Kyung Hee University Hospital at Gangdong, Seoul 134-727, South Korea
a r t i c l e
i n f o
Article history: Received 9 July 2012 Revised 7 August 2012 Accepted 10 August 2012 Available online 16 August 2012 Keywords: Molecular beacon FRET Protein quantitation Mucin 1
a b s t r a c t Mucin 1 (Muc1) is a glycoprotein expressed on most epithelial cell surfaces, which has been confirmed as a useful biomarker for the diagnosis of early cancers. In this study, we demonstrate that a quantum dot (QD)-aptamer beacon acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3, in the presence of the target molecules, Muc1. Release of intercalated BOBO-3s from the QD-conjugated aptamers results in a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission, allowing for label-free Muc1 detection and quantitation. We attain highly specific and wide-range detection (from 50 nM to 20 lM) of Muc1, suggesting that our QD-aptamer beacon can be a potential alternative to immuno-based assays for Muc1 detection. The detection methodology is expected to be improved for the early diagnosis of different types of epithelial cancers of large populations. Ó 2012 Elsevier Ltd. All rights reserved.
Mucins, cell-surface associated glycoproteins, are bound to cells by an integral transmembrane domain.1 Among the family, mucin 1 (Muc1) contains a hydrophobic membrane-spanning domain of 31 amino acids, a cytoplasmic domain of 69 amino acids, and an extracellular domain consisting of a region of nearly identical repeats of 20 amino acids per repeat.1 Muc1 has a protection function as its biological role by binding to pathogens and possibly functions in a signal transduction pathway.2 Muc1 is overexpressed in almost all human epithelial cell adenocarcinomas, including breast,3 gastric,4 colorectal,5 lung,6 prostate,7 ovarian,8 pancreatic,9 and bladder carcinomas.10 Moreover, the expression of Muc1 in these tissues usually lacks regular expression patterns, making the protein ubiquitously and randomly expressed all over the cell surface.11 Thus, large amounts of the protein can be found in blood of cancer patients,12 which allows serum assays for Muc1 to be potentially useful for early cancer detection. In this regard, several techniques including ELISA, dot blotting, Western blotting, immunohistochemistry and immunofluorescence have been developed for Muc1 detection and quantitation.13 In particular, two immune-based assays using anti-Muc1 antibodies have been recently developed to successfully monitor the concentration of Muc1 in serum.13 However, the production of a specific antibody for these antibody-based assays is time-consuming and not a fool proof procedure. In addition, the procedures usually use radioactively or fluorescently labeled secondary antibodies for sensitivity purposes. In the present study, we explore an aptamer-based detection methodology for Muc1 quantitation, with an ultimate goal of ⇑ Corresponding author. Tel.: +82 2 961 2186; fax: +82 2 966 3701.
E-mail address: [email protected] (S.S. Hah). These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.08.037
developing practical and quantitative analytical tools for tumor markers. Aptamers are a special class of nucleic acids that can specifically bind, with high affinity, to a wide array of target molecule.14 They have originated from large random-sequence nucleic acid libraries via an in vitro evolution process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment).15 To date, a wide range of DNA- and RNA-based aptamers have been discovered that bind with the dissociation constants ranging from nanomolar to picomolar level to metal ions,16 small molecules,17 proteins,14 and even entire cells.18 Importantly, aptamers rival antibodies, the traditional recognition molecule, because of a number of unique features of aptamers.14,19 First, the limit of having to use cell lines or animals, as is necessary for antibodies, can be overcome. Aptamers, once selected, can undergo subsequent amplification through polymerase chain reaction or transcription to produce a large quantity with high purity. Second, the simple chemical structure of aptamer makes it amendable to further modifications with functional groups based on the experimental purposes.7 Finally, aptamers are more stable than antibodies, allowing them to be suited for applications requiring harsh conditions (e.g., high temperature or extreme pH). Among a variety of aptamer-based alternatives to traditional immunoassays, molecular beacon assays are ideal for both in vitro and in vivo real-time and label-free protein sensing, because the beacon assay relies on an aptamer probe with a labeled fluorescent donor at one end and an acceptor, either a fluorophore or a quencher, at the other end,20 allowing fluorescence resonance energy transfer (FRET) between the donor and the acceptor to occur in the absence of the target. In the presence of the target, however, a binding-induced conformational change of the aptamer probe is initiated and alters the beacon’s donor– acceptor distance, leading to a change of FRET efficiency.21 Then
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S. Shin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6081–6084
Scheme 1. Label-free detection of Mucin 1 (Muc1) with beacons of QD-aptamer nanoconjugates loaded with BOBO-3s. Muc1 binding to the aptamer probes on QDs causes conformational change, resulting in dissociation of the pre-stained BOBO-3s from the QD-aptamer conjugates. Thus, Muc1 can be quantified by the increase QD emission at 565 nm and the decrease of FRET-mediated BOBO-3 emission at 605 nm.
Figure 1. (a) Fluorescence spectra of QD-aptamer nanoconjugates. (b) Fluorescence spectra of QD-aptamer nanoconjugates loaded with BOBO-3s. The intercalated dyes induce FRET-mediated decrease of QD fluorescence at 565 nm and increase of BOBO-3 fluorescence at 605 nm, which can be alternatively shown in (c) resulting from subtraction of the fluorescence intensities of QD-aptamer nanoconjugates loaded with the intercalating dyes from those of QD-aptamer nanoconjugates.
the target binding can be quantified based on the emission change at either donor’s or acceptor’s characteristic wavelength or both. An aptamer beacon, thus, has not only fluorescence sensitivity but also multi-dimensional specificity for label-free protein detection.
Whereas aptamers are used as a recognition element of the beacons, quantum dots (QDs) are used as a signal transducing element or the beacon’s FRET donor in the present study. QDs are semiconducting nanoparticles, which can be prepared with interesting optical properties.22 The fluorescent properties of QDs including phostostability, narrow emission bands, broad excitation spectra, and accessibility to versatile functionalization are the key advantages for their use as FRET donors,23 since QDs absorb light over a broad spectral range and fluoresce at wavelengths determined by their physical sizes, producing precise and narrow spectral emissions, and QDs are extremely efficient at absorbing light and converting it to a highly stable fluorescent emission.22 These niches have paved a new way to the development of QD-aptamer beacons.24 As the beacon’s FRET acceptors or reporters, we make use of a dimeric cyanine DNA intercalating dye BOBO-3 (1,10 -(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methylidene]-pyridinium tetraiodide, (excitation at 570 nm, emission at 605 nm),25 instead of additional reporter-labeled oligonucleotides to work with the aptamer probe. Our strategy is illustrated in Scheme 1. A single-stranded antiMuc1 aptamer probe is covalently conjugated to QDs (emission maxima at 565 nm).26 Then BOBO-3 that shows large fluorescence enhancement when intercalated into a double helix,27 is used to stain the duplex regions, leading to formation of QD-aptamer conjugates loaded with BOBO-3s, as clearly demonstrated in Figure 1. In the absence of the target protein, Muc1, a FRET-mediated emission of the intercalated BOBO-3s can be observed when QD is illuminated at 365 nm. Figure 1(b) shows that 365-nm excitation of the QD-aptamer nanoconjugates loaded with the intercalating dyes results in the decrease of QD fluorescence and the increase of BOBO-3 fluorescence, and that the energy transfer efficiency is estimated to be as high as 40%. Such a moderate energy transfer efficiency is attributed presumably to the fact that the thickness of the aptamer coating for dye staining (ca. 2.5 nm) is approximately a half of the Förster radius (57.7 Å) for the FRET pair of QDs and BOBO-3s.25 On the other hand, the presence of Muc1 which can bind to the aptamer, induces conformational change of the aptamer, resulting in dissociation of BOBO-3s from QD-aptamer conjugates. Thus, a decrease in the FRET-mediated BOBO-3 emission and an increase in the QD fluorescence are observed, which can allow for quantitation of the Muc1 concentration. Label-free quantitation of Muc1 in a clear buffer using our QDaptamer conjugates loaded with BOBO-3s is demonstrated in Figure 2. We measured the ratios of the emission intensities at 565 nm and 605 nm, respectively, and Figure 2(a) shows that the beacon in a buffer produces a 10–60% FRET signal change in response to 50 nM to 20 lM Muc1. It corresponds to a dynamic range across three orders of magnitude. The linear range is outlined in Figure 2(b), which demonstrates the achievement of 50 nM LOD (limit of detection).
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Figure 2. Label-free quantitation of Muc1 using our QD-aptamer nanoconjugates loaded with BOBO-3s. (a) Normalized ratios of the emission intensities of QDs at 565 nm as a function of Muc1 concentration, showing that the beacon in a buffer produces a 10–60% FRET signal change in response to 50 nM to 20 lM Muc1. The data from the experiment reveal that apparent dissociation constant (Kdapp) between the QD-conjugated aptamer and Muc1 is approximately 78 pM. (b) Logarithmic scale graph for the obtained data.
To summarize, a new QD-aptamer beacon, which is advantageous over the previously reported Muc1 sensing assays in the aspect of simplicity to generate the fluorescence change from QDaptamer conjugates in a target specific manner, has been developed by using a DNA intercalating dye, BOBO-3, as a FRET reporter. The beacon is prepared by pre-staining QD-aptamer nanoconjugates loaded with BOBO-3s and acts by Muc1 binding-induced dye displacement, which shows an increase in QD emission at 565 nm and a decrease in FRET-mediated dye emission at 605 nm. Accordingly, specific and label-free Muc1 sensing with a 50 nM to 20 lM dynamic range is achieved. Our results suggest that the QD-aptamer beacon can be a potential alternative to immuno-based assays for Muc1 detection, by bringing the power of QD fluorescence technology to a work-horse application in diagnostics. It should also be noted that because of the advantageous optical properties of QDs over conventional organic dyes and a wide versatility of aptamers for selection, our detection methodology can be applied in principle to other protein biomarkers, leading to improvement for the early diagnosis of different types of epithelial cancers of large populations. Acknowledgments This work was supported by Basic Science Program through the National Research Foundation of Korea (KRF) funded by the Ministry of Education, Science and Technology (MEST) (Nos. 20110021956 and 2012-001680). References and notes 1. (a) Gendler, S. J.; Spicer, A. P. Annu. Rev. Physiol. 1997, 57, 607; (b) Hollingsworth, M. A.; Swanson, B. J. Nat. Rev. Cancer 2004, 4, 45.
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QD concentrations were measured by optical absorbance, using extinction coefficients provided by the supplier. Sulfosuccinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, Sigma) was used 0 as cross-linker. HPLC-purified RNA-based probes (5 -HS-(CH2)6-GCGAG CGCAG
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TTGAT CCTTT GGATA CCCTG GGCTC GC-30 , Ref. 14b) for specific binding to Muc1 proteins were purchased from Bioneer (Daejeon, Korea), which was used for conjugation with the activated QDs following dithiothreitol treatment. The resulting QD-aptamer products were dissolved in PBS (200 lL) and stored at 4 °C for further experiments. Prior to detection of Muc1 (Peptron, Daejeon, Korea), each QD-aptamer (with 1 lM QD) nanoconjugate was pre-treated for annealing and mixed with 10 lM BOBO-3 (Invitrogen) in PBS in the dark for 1 h. The resulting QD-aptamer conjugates loaded with BOBO-3s were separated by centrifugation at 200g
for 1 min from the unbound BOBO-3s in the supernatant. Then, a given Muc1 sample dissolved in PBS was added with a final volume of 200 lL to the QDaptamer conjugates loaded with BOBO-3s. After 1-h incubation, the mixture was transferred to a fluorescence microplate reader, to measure its FRETmediated BOBO-3 emission signal (365-nm excitation, followed by 565- and 605-nm emission). 27. Ruedas-Rama, M. J.; Orte, A.; Crovetto, L.; Talavera, E. M.; Alvarez-Pez, J. M. J. Phys. Chem. B 2010, 1094, 114.