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Fluorescence Polarization Based Displacement Assay for the Determination of Small Molecules with Aptamers Jorge A. Cruz-Aguado and Gregory Penner* NeoVentures Biotechnology Inc., 700 Collip Circle, London, Ontario, N6G 4X8, Canada The conversion of an aptamer-target binding event into a detectable signal is an important step in the development of aptamer-based sensors. In this work, we show that the displacement of a fluorescently labeled oligo from the aptamer by the target can be detected by fluorescence polarization (FP). We used Ochratoxin A (OTA), a small organic molecule (MW ) 403) as a case study. A detection limit of 5 nM OTA was achieved. The method presented here provides an advantage over fluorophore-quenching systems and other steady-state fluorescence approaches in that no modification of the aptamer or the target is required. Additionally, the signal is produced by the displacement event itself, so no further agregation or conformational events have to be considered. This analytical method is particularly useful for small targets, as for large targets a direct measurement of the FP change of a labeled aptamer upon binding can be used to determine the concentration of the target. The results presented here demonstrate that aptamers and inexpensive labeled oligos can be used for rapid, sensitive, and specific determination of small molecules by means of FP. A number of aptamers have been identified that bind specifically to small molecules.1-3 This success with identification has not however been followed by a significant commercial application for the quantitative determination of target molecule concentration. One contributing factor to this lack of quantitative application has been due to implicit restrictions in the technologies employed to convert aptamer-target interactions into signals that provide adequate sensitivity. In this work, we show that aptamers can be used for the quantitative determination of target molecules by measuring the change in fluorescence polarization (FP) that a fluorescently labeled oligo undergoes when it is displaced from the aptamer by the target. FP is inversely proportional to the rate of rotation and tumbling of a molecule. A key factor influencing the FP value for any molecule or complex of molecules is the molar mass of the molecule being measured. The use of FP to measure aptamer binding to large protein * To whom correspondence should be addressed. E-mail: gpenner@ neoventures.ca. Phone: (519) 858 5052. Fax: (519) 858 5142. (1) Vianini, E.; Palumbo, M.; Gatto, B. Bioorg. Med. Chem. 2001, 9, 2543– 2548. (2) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J. Am. Chem. Soc. 2001, 123, 4928–4931. (3) Sazani, P. L.; Larralde, R.; Szostak, J. W. J. Am. Chem. Soc. 2004, 126, 8370–8371. 10.1021/ac8017058 CCC: $40.75 2008 American Chemical Society Published on Web 10/24/2008
molecules has been previously demonstrated with direct fluorescent labeling of the aptamer.4 To date though, this approach has been limited to the analysis of aptamer target interactions where the target is substantially larger than aptamer. In this study, we extend the use of FP determination of aptamer/target binding by measuring the FP change that takes place when a small fluorescently labeled oligo is displaced from an aptamer with a higher mass by an analyte. In this case, the FP values decrease as a function of the concentration of the analyte, and this change in FP can then be used to determine analyte concentration. Thus, the generation of the detection signal with this arrangement is independent of the mass of the analyte. Displacement assays have been shown to have a universal applicability in chemical and biochemical analysis; however, the generation of a signal has been limited primarily to fluorophore -quencher5 or colorimetric6,7 systems. Recently, Li and Ho5 reported a strategy based on the displacement of an annealed oligo from an aptamer as a method to determine ATP and thrombin concentration. Their strategy involved the generation of a detection signal through the incorporation of a fluorescent nucleotide analogue in an oligo that annealed to the aptamer. An increase in the quantum yield of the fluorescence was linearly related to the amount of oligo displaced by target molecule binding to the aptamer. As an alternative approach, they also designed an oligo that quenched itself by including both a fluorophore and a quencher on the oligo. The approach presented in the present work has the advantage that the aptamer does not have to be modified in any way to accommodate the detection system, in difference to previous work.2 Additionally, the signal does not depend on any conformational change within the liberated oligo or on the stacking interaction between the fluorophore and the aptamer bases; thus, the sensitivity and robustness of the detection method is improved. We demonstrate the effectiveness and simplicity of the method with an aptamer we developed for Ochratoxin A (OTA). OTA is a potent toxin produced by several species of Aspergillus and Penicillium that can be present in grains and other food products. Current approved methods for quantitative determination are limited to purification with an antibody-based affinity column followed by HPLC.8 A number of sensitive methods have been developed;9-11 however, each of these methods requires the use of sophisticated Jayasena, S. D. Clin. Chem. 1999, 45, 1628–1650. Li, N.; Ho, C. J. Am. Chem. Soc. 2008, 130, 2380–2381. Stojanovic, M. N.; Landry, D. W. J. Am. Chem. Soc. 2002, 124, 9678–9679. Nguyen, B. T.; Anslyn, E. V. Coord. Chem. Rev. 2006, 250, 3118–3127. NEOGEN Europe Ltd. NeoColumn for Ochratoxin A. Technical booklet, July 2004. (9) Aksenov, I; Eller, K. I.; Tutel’ian, V. A. Gig. Sanit. 2006, 4, 50–53.
(4) (5) (6) (7) (8)
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instrumentation. FP, on the other hand, is a relatively inexpensive technique that can be integrated into analysis with a hand-held devise.12 We identified a number of aptamers that bind with strong affinity and specificity to OTA (to be submitted). The present study shows that low nanomolar concentrations of OTA can be determined using the change in FP generated as result of the competition between OTA and short fluorescently labeled oligonucleotides for one of those aptamers. EXPERIMENTAL SECTION The ssDNA oligonucleotides were synthesized by Sigma-Genosys. The sequence of the OTA aptamer 1.12.2 was 5′-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3′. Antisense oligos labeled with fluorescein at the 5′-end were designed to anneal different sections of the aptamer. The sequences of the oligos from 5′ to 3′ were as follows: Fluo1.12.2-1, CAC CCG ATC; Fluo1.12.2-2, CGC CAC CCA; Fluo1.12.2-3, CTC CCT TTA; Fluo1.12.2-4, TGT CCG ATG. There was a 6-carbon saturated chain space arm between the fluorescein and the oligo. All buffer solutions were filtered through a 45-µm syringe filter to reduce the possibility of light scattering. The stock solutions of the oligo and the aptamer or the oligo alone were heated to 90 °C and incubated at room temperature for at least 0.5 h before use. For the assays, 10 µL of 200 nM aptamer and 200 nM oligo combined in the same buffer were added to 190 µL of target in buffer 10 mM TRIS pH 8.5, 120 mM NaCl, 20 mM CaCl2, and 5 mM KCl, for a final concentration of 10 nM, or 10 µL of 2 µM solution of each aptamer and oligo was added for a final concentration of 100 nM each, in Corning 96-well nonbinding surface black microplates. The solutions were mixed with the pipet in the microplate to ensure good mixing and measured immediately. The time between mixing the solutions in the microplate and the measurement was no more than 30 s. Further FP measurements were taken after time intervals of 30, 60, and 120 min after the initiation of incubation at room temperature with no shaking. The sensitivity of the assay was determined as the concentration of the analyte that produced a signal higher than three times the SD of the solution in the absence of OTA. FP measurements were made in three independent experiments using a Tecan Safire II monochromator microplate reader (Tecan). Excitation light was generated with an LED lamp with an excitation wavelength maximum of 470 nm. The emission wavelength was set at 525 nm and the emission bandwidth at 12 nm. The G factor was determined from a standard solution of fluorescein in a 0.2 M carbonate buffer pH 9.6 (FP ) 20 mP); 30 readings were collected in each measurement. Preliminary measurements with plain buffer as well as with standard solutions of fluorescein in the same buffer used for the assays showed a lack of scattering, as the FP values of free fluorescein were close to 20 mP. In all the experiments, the ratio of fluorescence intensity signal/background was higher than 1000 for both parallel and perpendicular configurations. All measurements were corrected for the background fluorescence and the G factor by the instrument software, X-Fluor. (10) Koeller, G.; Wichmann, G.; Rolle-Kampczyk, U.; Popp, P.; Herbarth, O. J. Chromatogr., B 2006, 840, 94–98. (11) Jimenez, A. M.; Lo´pede Cerain, A.; Gonzalez-Pen ˜as, E.; Bello, J. Chromatography 1999, 50, 457–460. (12) Gryczynski, Z.; Abugo, O. O.; Lakowicz, J. R. Anal. Biochem. 1999, 273, 204–211.
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Figure 1. Fluorescence polarization of 100 nM solution of 9-mer oligos (black bars), the same oligos in the presence of 100 nM OTA aptamer (gray bars), and the oligos in the presence of the aptamer and 1 µM OTA (white bars).
RESULTS AND DISCUSSION In order to find oligos that anneal to the OTA aptamer and that are displaced by the target, we designed a series of 9-mer oligos complementary to different sections of an aptamer that binds OTA with a Kd ) 50 nM. The addition of the oligos Fluo1.12.2-2 and Fluo1.12.2-4 to the aptamer in the absence of OTA resulted in the largest increase in FP values (Figure 1). This indicates that they annealed to the aptamer at room temperature. The increase in FP is expected as the molecular mass of the complex oligo-aptamer is five times higher than the oligo alone. The oligos were designed such that the melting temperature between themselves and the aptamer was slightly higher than room temperature. We used DINAmelt to estimate the proportion of oligo annealed to the aptamer at room temperature.13 At 20 °C, the proportion of oligonucleotide bound to aptamer at equilibrium was expected to be Fluo1.12.2-1 43%, Fluo1.12.2-2 90%, Fluo1.12.2-3 7%, and Fluo1.12.2-4 50%. These values correspond reasonably well to the increases detected in respective FP values. In the presence of 1 µM OTA, the FP values of the Fluo1.12.2-4/aptamer complex exhibited the largest decrease between free and annealed oligo (Figure 1.). To determine whether the displacement of Fluo1.12.2-4 by OTA was proportional to the concentration of OTA in solution, we measured the FP values generated in the presence of different concentrations of OTA. Steady-state FP is the mean value of FP from all the fluorescently labeled molecules. This implies that the concentrations of the aptamer and the oligo in the assay should be close to the expected range of concentrations of the analyte in order to obtain maximum levels of sensitivity. At the same time, the sensitivity could be limited by the instrumental detection limit for fluorescein fluorescence intensity; however, this last factor was not relevant in our experiments, as the fluorescence intensity in our samples was well above the detection limit. When we used concentrations of aptamer and oligo of 10 nM each, we obtained a sensitivity of 5 nM OTA, which corresponds to 1 pmol in the detection volume of 200 µL. Even though the sensitivity of this approach does not match the sensitivity achieved by other methods of determination of ochratoxin A,9-11 this level of sensitivity was sufficient to determine concentrations of the toxin that exceed regulatory requirements.14 The sensitivity achieved with this approach provides a solid basis for a generic platform technology. With aptamer and oligo concentrations set at 10 nM each, the regression of FP values with OTA concentra(13) Markham, N. R.; Zuker, M. Nucleic Acids Res. 2005, 33, W577-W581.
Figure 2. Effect of concentration of OTA on the FP of a solution containing an aptamer (10 nM) and the oligo Fluo1.12.2-4 (10 nM). Inset: absolute change in FP at low OTA concentrations. Measurements were made less than 30 s after mixing the solutions. Each point is the mean of three experiments, error bars are the SD. Figure 4. Effect of NAP (1 µM) and warfarin (1 µM) on the detection of OTA (100 nM) with a fluorescence polarization based displacement assay using 10 nM solutions of aptamer (A) and oligo Fluo1.12.2-4 (O). Each point is the mean of three experiments, error bars are the SD.
Figure 3. Effect of concentration of OTA on the FP of a solution containing an aptamer (100 nM) and the oligo Fluo1.12.2-4 (100 nM). Measurements were made less than 30 s after mixing the solutions. Error bars are the SD.
tion up to 12 nM OTA (Figure 2) was linear (y ) 0.53x; r2 ) 0.97). When the concentrations of aptamer and oligo were increased to 100 nM, the linearity of the FP response was extended up to 200 nM OTA (y ) -0.086 + 125.3x; r2 ) 0.99) with a concurrent loss of sensitivity below 12 nM (Figure 3). The sensitivity of this method corresponds to a concentration of OTA 10 times lower than the Kd of the aptamer-OTA complex. This indicates that the binding events were effectively transformed into a detectable signal as the binding of as low as 5% of the aptamer to the target produced a significant change in FP. It is possible that further improvement in sensitivity could be achieved by reducing the space arm between the fluorophore and the oligo. In this way, the local rotation of the fluorophore in the molecule would have a lower weight in the total FP, and thus the FP values would be more dependent on the molecular mass. The method presented here enabled immediate measurement of target concentration as equilibrium between aptamer/competitor and target molecule occurs in less than 30 s (the time taken to perform FP measurements). The signal was steady up to 1 h after the first measurement (data not shown), after which the linearity was lost, presumably because of evaporation of the solutions and concomitant changes in concentration. The rapid nature of the assay is of particular importance in field applications where individual samples must be analyzed in a short time. In contrast to current laboratory methods of determination of OTA and other mycotoxins, FP is a relatively inexpensive technique that can be adapted to handheld instruments.12 Hence, the FP displacement assay presented in this work could enable the chemical analysis under field conditions of any molecule for which an aptamer is identified. In order to determine the specificity of the method, we tested the effect of the presence in the assay solution of molecules that
have structural similarities with OTA. We used N-acetylphenylalanine, a molecule with a structure that is represented completely as a part of OTA, and warfarin. The similarity between warfarin and OTA is emphasized by the fact that they occupy the same binding site in human serum albumin, the main carrier of OTA in the body under natural exposure.15 None of these compounds exhibited a significant effect on the FP of the aptamer-oligo complex or on the change in FP produced by OTA even when they were present in the solution at concentrations 10 times higher than the concentration of the target (Figure 4). This demonstrates the specificity of the analytical system presented here. Neither ethanol (10%) nor dimethyl sulfoxide (5%), solvents that could be present in some target solution samples, affected the accuracy of the method (see Supporting Information). CONCLUSIONS This report describes a novel strategy for the use of aptamers in quantitative analysis. The FP technique presented here measures a signal that is neither influenced by the detection system nor dependent on conformational changes of the aptamer or the displaced oligo. In principle, this method could enable the detection of any molecule for which an aptamer is identified. In contrast to other recently published aptamer based analysis techniques,16 this method does not require sophisticated instruments and could be adapted to hand-held devices. Further improvements to the method are possible through the identification of aptamers with stronger target affinity and increasing the FP response to the molecular mass of the complex. ACKNOWLEDGMENT Financial support for this work was provided by the Ontario and Canadian governments, as well as from the Canadian Wheat Board. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text.. This material is available free of charge via the Internet at http://pubs.acs.org Received for review August 13, 2008. Accepted October 2, 2008. AC8017058 (14) O’Brien, E.; Dietrich, D. R. Crit. Rev. Toxicol. 2005, 35, 33–60. (15) Il’ichev, Y. V.; Perry, J. L.; Simon, J D J. Phys. Chem. B 2002, 106, 452– 459. (16) Wang, J.; Zhou, H. S. Anal. Chem. 2008, 80, 7174–7178.
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