Luminescence Switching by Hybridization-Directed Mixed Lanthanide

Dec 17, 2009 - ... University of Turku, Tykistökatu 6 A 6th floor, FI-20520 Turku, Finland. We have ... used as probes to detect a complementary targ...
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Anal. Chem. 2010, 82, 751–754

Luminescence Switching by Hybridization-Directed Mixed Lanthanide Complex Formation Ulla Karhunen,* Lumi Jaakkola, Qi Wang, Urpo Lamminma¨ki, and Tero Soukka Department of Biotechnology, University of Turku, Tykisto¨katu 6 A 6th floor, FI-20520 Turku, Finland We have developed a homogeneous assay method in which the lanthanide ion carrier and light absorbing components of a luminescent lanthanide chelate are separated in two distinct molecules that can together form a luminescent mixed chelate complex. The separated label moieties were conjugated to oligonucleotides which were used as probes to detect a complementary target DNA. The background signal of the assay was very low, indicating the signal was highly dependent on the hybridization of the two probes on adjacent positions on the target oligonucleotide. Lanthanide chelates and cryptates, due to their enhanced detectability compared to traditional organic fluorophores, are widely used labels in bioanalytical applications. The lanthanide ions themselves have a poor absorptivity and, therefore, require an organic chromophore coupled to a chelating ligand to elicit the exceptional luminescence properties including long emission lifetime, large Stokes’ shift, and the characteristic narrow-banded emission profile. Together with time-resolved fluorometry, they allow efficient elimination of the background fluorescence from biological material and, thus, increase the potential sensitivity.1 Lanthanide chelates have also been used as donors in distancedependent resonance energy transfer2 assays. Unfortunately, these homogeneous assays have some principal limitations: the donor cross talk at the measurement wavelength and the reabsorption of the donor emission by the acceptor inevitably increase the fluorescence background and limit the dynamic range and performance obtained. The aim of our study was to explore the possibility of a homogeneous assay that could avoid the limitations of resonance energy transfer assays and in which the signal generation would be highly specific and the background signal minimal. It was reasoned that this could be achieved by splitting the intrinsically luminescent lanthanide chelate into two distinct components: a nonluminescent ion carrier chelate and a light harvesting antenna ligand. The lanthanide ion, EuIII, is strongly coordinated to the carrier ligand by six or seven teeth forming the ion carrier chelate and leaving three or two coordination sites, respectively, to be occupied by quenching water molecules and to be replaced with a complementing tridentate antenna ligand. Recently, self-assembled luminescent lanthanide complexes have been presented resulting from the enforced, close proxim* To whom correspondence should be addressed. Tel: (+358)2 333 8084. Fax: (+358)2 333 8050. E-mail: [email protected]. (1) Hemmila¨, I.; Mukkala, V.-M. Crit. Rev. Clin. Lab. Sci. 2001, 38, 441–519. (2) Mathis, G. Clin. Chem. 1993, 39, 1953–1959. 10.1021/ac9020825  2010 American Chemical Society Published on Web 12/17/2009

Figure 1. Schematic illustration of the oligonucleotide hybridization directed lanthanide chelate complementation assay. (a) Without target oligonucleotide, the probe A-EuIII-N1 and the probe B-antenna are nonluminescent. (b) Target oligonucleotide induces the formation of the highly luminescent mixed chelate complex, which is excited at 340 nm, and the long-lifetime luminescence emission is measured at 615 nm.

ity of a sensitizer and EuIII complex immobilized on a glass surface3 and the use of an intrinsic tryptophan residue in a protein to sensitize TbIII luminescence of a bound peptide.4 A homogeneous hybridization assay by the use of DNA-template mediated formation of a luminescent lanthanide complex was originally presented by Oser and Valet, but the modulation they obtained was less than 3-fold.5 The principle of the hybridization assay method based on lanthanide chelate complementation is presented in Figure 1. Two 16-mer oligonucleotide probes, probe A labeled with a lanthanide ion carrier chelate [N1-(4-isothiocyanatobenzyl)diethylenetriamineN1,N2,N3,N3-tetrakis(acetato)europium(III)]6 (EuIII-N1, 2 in Figure 2) at an amino-modified thymine placed one nucleotide internal to the 3′ end and probe B labeled with a light-harvesting antenna ligand, 4-((isothiocyanatophenyl)ethynyl)pyridine-2,6-dicarboxylic acid (antenna, 1 in Figure 2) at an amino-modified thymine placed one nucleotide internal to the 5′ end, were hybridized to adjacent positions on a complementary 32-mer target oligonucleotide. The label moieties were positioned at one nucleotide distance from the adjacent termini based on our preliminary optimization experiments. The intermolecular binding affinity of probe A-EuIII-N1 and probe B-antenna with each other is minimal and no luminescence is observed in the absence of a complementary target oligonucleotide. In the presence of the (3) Hsu, S.-H.; Yilmaz, M. D.; Blum, C.; Subramaniam, V.; Reinhoudt, D. N.; Velders, A. H.; Huskens, J. J. Am. Chem. Soc. 2009, 131, 12567–12569. (4) Pazos, E.; Torrecilla, D.; Va´zquez Lo´pez, M.; Castedo, L.; Mascaren ˜as, J. L.; Vidal, A.; Va´zquez, M. E. J. Am. Chem. Soc. 2008, 130, 9652–9653. (5) Oser, A.; Valet, G. Angew. Chem., Int. Ed. Engl. 1990, 29, 1167–1169. (6) Mukkala, V.-M.; Mikola, H.; Hemmila¨, I. Anal. Biochem. 1989, 176, 319– 325.

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Figure 2. Structures of the light harvesting antenna ligand 4-((4isothiocyanatophenyl)ethynyl)pyridine-2,6-dicarboxylic acid (antenna, 1) and the lanthanide ion carrier chelate [N1-(4-isothiocyanatobenzyl)diethylenetriamine-N1,N2,N3,N3-tetrakis(acetato)europium(III)] (EuIII-N1, 2).

complementary target oligonucleotide, the probe A-EuIII-N1 and probe B-antenna hybridize simultaneously onto the target, and EuIII-N1 and antenna form a long-lifetime luminescent mixed chelate complex. EXPERIMENTAL SECTION Reagents. Oligonucleotides (probe A 5′-CATTGCTACGATCC(C6dT)C-3′, probe B 5′-T(C2dT)CCTGCTACTGCATC-3′, and target oligonucleotide 5′-GATGCAGTAGCAGGAAGAGGATCGTAGCAATG-3′) were purchased from Aldrich (St. Louis, MI), and the noncomplementary target oligonucleotide (5′- CTGCTCTATCCACGGCGCCCGCGGCTCCTCTC-3′) was purchased from Biomers.net (Ulm, Germany). The homogeneous assays were performed by the use of low fluorescence 96-well Maxisorp microtitration plates purchased from Nunc (Roskilde, Denmark). All dilutions were made in assay buffer containing 50 mM Tris-HCl (pH 7.75), 600 mM NaCl, 0.1% (v/v) Tween 20, 0.05% (w/v) NaN3, 30 µM diethylenetriaminepentaacetic acid (DTPA). Safety Considerations. All reagents of the organic syntheses should be handled with caution. Thiophosgene is toxic by inhalation, and mechanical ventilation should be used. Skin and eye contact and inhalation should be avoided with the rest of the reagents as well. Synthesis of Light Harvesting Antenna. The novel light harvesting antenna was prepared according to the steps shown in Scheme 1, as described below. Diethyl 4-((4-aminophenyl)ethynyl)pyridine-2,6-dicarboxylate (4). Diethyl 4-bromopyridine-2,6-dicarboxylate (3)7 (602 mg, 2 mmol), 4-ethynylaniline (300 mg, 2.5 mmol), Pd(PPh3)2Cl2 (56 mg, 0.08 mmol), CuI (30.4 mg, 0.16 mmol), tetrahydrofuran (8 mL), and triethylamine (7 mL) were mixed in a round flask. The suspension was stirred at 55 °C under argon for 12 h. All volatiles were evaporated under vacuum, and the residue was purified by silica gel chromatography (methanol/dichloromethane 0-1%) to give 500 mg (yield 74%) of the product. Sodium 4-((4-aminophenyl)ethynyl)pyridine-2,6-dicarboxylate (5). A solution of 4 (230 mg, 0.68 mmol) and NaOH (0.12 g, 3 mmol) in ethanol (26 mL) and water (20 mL) was stirred at 35 °C for 3 h. After removal of all solvents, the residue was washed twice with 20 mL of 95% ethanol and filtrated. The product (5) was dried in vacuum and used for the next reaction without further purification. 4-((4-Isothiocyanatophenyl)ethynyl)pyridine-2,6-dicarboxylic acid (1). Compound 5 (222 mg, 0.68 mmol) was placed in a flask and dissolved in water (30 mL). Chloroform (30 mL) and sodium (7) Takalo, H.; Kankare, J. Acta Chem. Scand. 1987, 41B, 219–221.

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bicarbonate (335 mg, 4 mmol) were added. Thiophosgene (230 µL, 3 mmol) was added, followed by vigorous stirring for 3 h. The precipitate was collected by filtration, washed with water, and dried in vacuum to give 220 mg of product (1). Labeling of Oligonucleotides. Probe A was labeled with the nonluminescent ion carrier chelate (EuIII-N1) and with intrinsically luminescent [2,2′,2′′,2′′′-[[4-[(4-isothiocyanatophenyl)ethynyl]pyridine-2,6-diyl]bis(methylenenitrilo)]tetrakis(acetato)europium(III)]8 (EuIII-7d, 6 in Figure 3), and probe B was labeled with the light absorbing antenna ligand 4-((isothiocyanatophenyl)ethynyl)pyridine-2,6-dicarboxylic acid (antenna) at the aminomodified thymines. Probe A, 25 nmol or 5 nmol, was incubated with a 20-fold molar excess of EuIII-N1 or EuIII-7d, respectively, in 50 mM carbonate buffer, pH 9.8, at +37 °C overnight. The total volume of the labeling reactions was 50 µL. For labeling of 50 nmol of probe B with antenna, the antenna was dissolved in N,N-dimethylformamide (Sigma-Aldrich) and combined with oligonucleotide dissolved in water, and thereafter, carbonate buffer, pH 9.8, was added to a concentration of 50 mM. In the labeling reaction, the molar excess of the antenna was 50-fold in a total volume of 110 µL. The reaction was incubated at +50 °C with slow rotation overnight. The purification of labeled probes was carried out with HPLC (instrumentation from Thermo Electron Corp., Waltham, MA) using an ODS C18 Hypersil column from Thermo Scientific (Waltham, MA) for purification of antenna-labeled probe B and Luna C18 (2) column from Phenomenex (Torrance, CA) for purification of EuIII-N1 and EuIII-7d-labeled probe A. Purifications were performed using a gradient from 86% A and 14% B to 70% A and 30% B in 21 min with a flow rate of 0.5 mL min-1 (A, aqueous 50 mM triethylammonium acetate (TEAA; Fluka Biochemica, Buchs, Switzerland); B, 50 mM TEAA in acetonitrile (J.T. Baker, Phillipsburg, NJ)). The liquid from the collected fractions was evaporated in vacuum (Hetovac VR-1, Heto-Holten A/S, Allerod, Denmark) and then dissolved again in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl. Labeled probes were characterized by measuring absorbance readings at 260 and 330 nm, and the total EuIII concentrations were measured with DELFIA system (PerkinElmer Life and Analytical Sciences, Wallac, Turku, Finland). Homogeneous Chelate Complementation Assay for Target Oligonucleotide. The total reaction volume in the homogeneous target oligonucleotide titration assay was 60 µL, containing the following reagent concentrations. Into the low fluorescence microtitration wells, 30 µL of assay buffer containing 20 or 100 nM probe A-EuIII-N1 and probe B-antenna was added, and thereafter, 30 µL of assay buffer containing 0-100 nM complementary target oligonucleotide or 4 or 20 nM noncomplementary target oligonucleotide was added. The plate was incubated at room temperature first at slow shaking for a short period of time and then without shaking for 15 min. Time-resolved fluorescence was measured with a 1420 Victor Multilabel Counter (PerkinElmer Life And Analytical Life Sciences, Turku, Finland) using a 340 nm excitation filter, 615 nm emission filter, 0.4 ms delay, and 0.4 ms measurement time. In addition to the (8) Takalo, H.; Mukkala, V.-M.; Mikola, H.; Liitti, P.; Hemmila¨, I. Bioconjugate Chem. 1994, 5, 278–282.

Scheme 1. Synthesis of the Light Harvesting Antenna Liganda

a i, PBr3 + Br2, 90°C; ii, EtOH; iii, 4-ethynylaniline, Pd(PPh3)2Cl2, CuI, THF, Et3N, 55°C; iv, NaOH, EtOH-H2O; v, thiophosgen, CHCl3, H2O, NaHCO3.

Figure 3. Structure of the intrinsically luminescent europium(III) chelate [2,2′,2′′,2′′′-[[4-[(4-isothiocyanatophenyl)ethynyl]pyridine-2,6diyl]bis(methylenenitrilo)]tetrakis(acetato)europium(III)] (EuIII-7d, 6).

15 min of incubation, a similar measurement was made in the assay at a time point of 1 h. Luminescence Spectra and Emission Lifetimes. Luminescence emission spectrum (using 325 nm excitation with 20 nm slit, 550-750 nm emission with 5 nm slit, 0.1 ms delay and 0.4 ms measurement time) and emission lifetime (using 325 nm excitation with 20 nm slit, 615 emission with 20 nm slit, 0.05 ms delay and 0.05 ms gate) of the target oligonucleotide directed complex of the probe A-EuIII-N1 and probe B-antenna and of the intrinsically luminescent probe A-EuIII-7d were measured with a Varian Cary Eclipse fluorescence spectrophotometer (Varian Scientific Instruments, Mulgrave, Australia). The target oligonucleotide (0 or 10 nM) was mixed with probe A-EuIII-N1 and probe B-antenna (50 nM) in assay buffer and incubated for 30 min at RT before the measurement. To compare the luminescence properties of the oligonucleotide directed complex to the luminescence properties of the intrinsically luminescent EuIII-chelate, the probe A-EuIII-7d was diluted to a concentration of 50 nM in assay buffer and measured. RESULTS AND DISCUSSION Homogeneous Chelate Complementation Assay for Target Oligonucleotide. The detection limit of the hybridization assay was 22 pM (1.3 fmol per assay) when the concentration of both probe A-EuIII-N1 and probe B-antenna was 50 nM (Figure 4). The detection limit was defined as the concentration corresponding to 1.2× background signal (employed because the observed coefficient of variation of the background signal was less than 2%). Further, addition of a noncomplementary 32-mer oligonucleotide (2 and 10 nM) resulted in no change in luminescence, indicating the signal was highly dependent on the simultaneous hybridization of the labeled probes to the target oligonucleotide, defined by the nucleotide sequences. To prevent the interaction of antenna ligand with trace amounts of uncomplexed EuIII, potentially dissociated from the ion carrier chelate, we included a 30 µM

Figure 4. Time-resolved fluorescence measured after hybridization of the antenna and EuIII carrier chelate labeled probe pair (10 nM: 0; 50 nM: O) with increasing concentration of the target oligonucleotide. cts: counts.

concentration of DTPA in the assay. With a thermodynamically more stable ion carrier chelate structure, the addition of DTPA in the assay could be avoided. Due to the addition of DTPA and because of improved design of a light absorbing antenna, the detection limit was better than previously described5 and the obtained signal to background ratio (1400:1) was outstanding when compared to the less than 3-fold change that Wang and others9 have reported and to the 30-fold ratio of Sueda and others10 in their luminescence-resonance energy transfer assay. The dynamic range of the assay covered 4 orders of magnitude, and the luminescence signal was stable for at least 1 h. Luminescence Spectra and Emission Lifetimes. The complex that formed in the presence of 10 nM target oligonucleotide from 50 nM probe A-EuIII-N1 and probe B-antenna generated luminescence spectrum (Figure 5a) similar to the intrinsically luminescent probe A-EuIII-7d (Figure 5b) with the main emission peak at 615 nm. In the absence of the target oligonucleotide, no luminescence emission with probe A-EuIII-N1 and probe B-antenna was detected. The luminescence decay time (τ) of the complex formed of probe A-EuIII-N1 and probe B-antenna in the presence of 10 nM target oligonucleotide was 618 µs (decay spectrum inset in Figure 5a), and τ of the probe A-EuIII(9) Wang, G.; Yuan, J.; Matsumoto, K.; Hu, Z. Anal. Biochem. 2001, 299, 169– 172. (10) Sueda, S.; Yan, J.; Matsumoto, K. Bioconjugate Chem. 2002, 13, 200–205.

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Figure 5. Luminescence emission spectrum of (a) oligonucleotide directed complex formed of probe A-EuIII-N1 and probe B-antenna with 0 nM (narrow line) and 10 nM (wide line) target oligonucleotide and (b) probe A-EuIII-7d. The fitted emission decay spectrum of the complex formed of probe A-EuIII-N1 and probe B-antenna with 10 nM target oligonucleotide and of the probe A-EuIII-7d are presented in figure insets.

7d was 380 µs (inset in Figure 5b). Excitation maximum of the mixed complex was at 325 nm, and efficiency at 340 nm was 87% of the maximum. CONCLUSIONS As a conclusion, the lanthanide chelate complementation-based homogeneous assay technology provides highly sensitive and quantitative detection of nucleic acids over a very wide dynamic range and may also be applicable to protein analysis. The proximity probe-based complete luminescence switching illustrated here is an uncomplicated alternative to the proximity ligation-based detection principle,11 in which a complex enzymatic procedure is employed for detection. The degree of luminescence signal modulation obtained by the chelate complementation is truly exceptional among homogeneous fluorescence-based methods. The signal generation is also highly specific, as the selfassembly of the mixed chelate complex is strictly controlled by simultaneous and precise binding of the two probes (one labeled with ion carrier chelate and the other with light-harvesting antenna) onto adjacent positions in the target oligonucleotide. The signal level in absence of target and in presence of noncomple(11) Fredriksson, S.; Gullberg, M.; Jarvius, J.; Olsson, C.; Pietras, K.; Gu´stafs¨ stman, A.; Landegren, U. Nat. Biotechnol. 2002, 20, 473– do´ttir, S. M.; O 477.

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mentary target were the same, demonstrating further the specificity and hybridization-dependency of the mixed chelate formation. The long-lifetime emission enables time-resolved fluorometry, which eliminates the problems due to autofluorescence commonly observed with biological samples. The method is possibly compatible with other lanthanides, since SmIII may be excited with the same light absorbing antenna structure utilized for EuIII, and an antenna suitable for excitation of TbIII and DyIII can also be designed. The unique emission bands of different luminescent lanthanide ions may further allow extension of the method to monitor several analytes simultaneously. ACKNOWLEDGMENT This work was supported by the Academy of Finland (Grant Number 119497). SUPPORTING INFORMATION AVAILABLE The NMR spectra of compounds 4 and 5, and exact mass of 1, 4, and 5. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review September 16, 2009. Accepted December 1, 2009. AC9020825