Catalytic Beacons for the Detection of DNA and Telomerase Activity Yi Xiao, ² Valeri Pavlov, ² Tamara Niazov, ² Arnon Dishon, ‡ Moshe Kotler, ‡ and Itamar Willner* ,² The Institute of Chemistry, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel, and Department of Pathology, The Hebrew UniVersity-Hadassah Medical School, Jerusalem 91120, Israel Received December 21, 2003; E-mail: willnea@vms.huji.ac.il The discovery of catalytic RNAs (ribozymes) sparked scientific interest directed to the preparation of new biocatalysts. 1,2 Analogous deoxyribozymes (catalytic DNAzymes) are not available in nature, but have been demonstrated synthetically. 3,4 An interesting DNAzyme that revealed peroxidase-like activities is a complex between hemin and a single-stranded guanine-rich nucleic acid (aptamer). 5 This complex catalyzed the oxidation of 2,2′-azino-bis(3-ethylbenzthi- azoline)-6-sulfonic acid, ABTS, by H 2 O 2 . It was suggested 6 that the intercalation of hemin into the complex results in the formation of the biocatalyst. We have shown that the hemin/G-quadruplex also mimics peroxidase by the generation of chemiluminescence in the presence of H 2 O 2 and luminol. 7 The use of DNAzymes as catalytic labels for biosensing is attractive since nonspecific adsorption processes associated with protein-based labels are eliminated. Nucleic acid beacons are extensively used as specific DNA sensing matrixes. The specific linkage of photoactive chromophores/ quenchers to the hairpin termini results in the chromophore luminescence quenching. The subsequent lighting-up of the chro- mophore luminescence by the hybridization of DNA with the hairpin was used as a general motif for the photonic detection of DNA. 8 The quenching of dyes by molecular or nanoparticle quenchers 9 or the fluorescence resonance energy transfer (FRET) between dyes was used for the optical detection of the hybridization of the DNA to the beacon. 8 Recently, the labeling of the beacon termini with redox-active units led to the electrochemical detection of hybridization to the hairpins. 10 The development of catalytic beacons may provide a major advance in DNA sensing, and recently, efforts to apply beacon structures for the catalyzed sensing of the hybridization were reported. 11 Also, catalytic DNA coupled to gold nanoparticles was reported as a colorimetric sensor for lead ions. 12 Here we report on the tailoring of catalytic beacons for the sensing of DNA and telomerase activity originating from HeLa cancer cells. We design hairpin structures that upon opening yield, in the presence of hemin, DNAzymes that allow the biocatalytic detection of the hybridization process. Scheme 1A depicts the method for applying the beacon (1) as a catalytic unit for the sensing of DNA (2). 13 The hairpin structure of (1) includes the sequence consisting of segments A and B that in an open configuration form the G-quadruplex with hemin that exhibits peroxidase-like activity. Since segment B is hybridized in the hairpin structure, the formation of the catalytic DNAzyme is prohibited. Hybridization of DNA (2) with the hairpin opens the beacon, and the released sequence (components A and B) self- assembles with hemin to form the catalytic DNAzyme that oxidizes ABTS (3) to the colored product (4) by H 2 O 2 . The hybridization and hairpin opening is detected spectroscopically by following the accumulation of (4) at λ ) 414 nm (ǫ ) 3.6 × 10 4 M -1 cm -1 ). Figure 1, curve a, shows the time-dependent color evolution upon the analysis of DNA (2) 4.3 μM. Knowing the activity of the pure DNAzyme, we estimate that 85% of the beacon was opened. The control experiment, curve c, follows the spectral changes of the hairpin (1) in the presence of hemin, H 2 O 2 , and ABTS and does not lead to any development of a color. Also, the hybridization of (2) with a hairpin structure that lacks the B segment in the “hairpin ² The Institute of Chemistry. ‡ Department of Pathology. Figure 1. Absorbance changes originating from the formation of (4) upon analysis of: (a) (2), 4.3 μM. (b) Absorbance generated by hemin and (2), 4.3 μM, in the absence of (1). (c) Color formed by hemin and (1) without (2). (d)-(h) Analysis of variable concentrations of (2) corresponding to 3.0, 2.15, 1.30, 0.40, and 0.2 μM, respectively. (i) and (j) The analysis of the SNP mutations (2a) or (2b), 4.3 μM. Experiments were performed in the presence of (1), 0.43 μM, hemin, 0.43 μM, ABTS, 3.2 mM and H 2O2, 3.2 mM in a 0.1 M Tris buffer solution, pH ) 8.1, that included MgCl2, 20 mM. Inset: Calibration curve corresponding to absorbance upon analyzing variable concentrations of (2) after a fixed time interval of 3 min. Scheme 1. (A) Analysis of DNA by Opening of a Beacon Nucleic Acid and the Generation of a DNAzyme. (B) Analyzing Telomerase Activity by a Functional DNA Beacon that Self-Generates a DNAzyme Published on Web 05/28/2004 7430 9 J. AM. CHEM. SOC. 2004, 126, 7430-7431 10.1021/ja031875r CCC: $27.50 © 2004 American Chemical Society