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Measurement of (aptamer – small target) KD using the competition between fluorescently labeled and unlabeled target and the detection of fluorescence anisotropy Alexey V. Samokhvalov, Irina V. Safenkova, Sergei Alexandrovich Eremin, Anatoly V. Zherdev, and Boris B. Dzantiev Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01699 • Publication Date (Web): 01 Jul 2018 Downloaded from http://pubs.acs.org on July 1, 2018
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Analytical Chemistry
Measurement of (aptamer – small target) KD using the competition between fluorescently labeled and unlabeled target and the detection of fluorescence anisotropy Alexey V. Samokhvalov1, Irina V. Safenkova1, Sergei A. Eremin2, Anatoly V. Zherdev1, Boris B. Dzantiev1* 1
A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia 2 Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia ABSTRACT: Registration of fluorescence anisotropy (FA) allows for characterizing the interactions of ligands with aptamers and other receptors under homogeneous conditions without reagents immobilization, prolonged incubations and products separation. We proposed approach for aptamer’s affinity determination by FA taking into account the difference in label fluorescence before and after complexation. The detailed step by step scheme using a native and fluorescently labeled ligand was described and justified in the paper. The scheme ensures the exclusion of data with low reliability and establishes valid criteria for selecting optimal concentrations of reagents (labeled ligand and aptamer) used in the experiments. The approach was experimentally tested using ochratoxin A (OTA), its fluorescein-labeled derivative (OTA-Flu), and the aptamer binding them. We demonstrated that it allows minimizing the influence of fluorescence change to accurately determine the dissociation constant. Based on FA registration, the binding constants of the aptamer–OTA-Flu and the aptamer–OTA complexes were found to be equal 245+33 and 63+18 nM, respectively. The value for the aptamer–OTA complexes was confirmed by the equilibrium dialysis technique. The resulting constant was 80±9 nM. The versatility and methodological simplicity of the proposed protocol, as well as the short implementation time, are why it can be recommended as an effective tool for characterizing aptamer-ligand complexes.
Aptamers are short, single-stranded nucleic acids capable of selectively binding molecular targets due to the presence of specific three-dimensional structures.1,2 The main method of obtaining new aptamers is systematic evolution of ligands by exponential enrichment (SELEX), which is the in vitro selection of oligonucleotides from libraries containing up to 1014 variants.3,4 The selected aptamers are then produced by chemical synthesis. The main advantages of aptamers as receptor molecules include the simplicity of their synthesis and modification with additional functional groups, their applicability in a wide range of conditions, and effective renaturation.5,6 These advantages lead to active use of aptamers in analytical systems7,8 and for targeted delivery of pharmaceuticals.9 The aptamer is largely determined by its affinity to the target ligand (the equilibrium dissociation constant of the complex, KD). Therefore, correct and easy-touse methods of measuring KD are in extreme demand.10 To measure the constants of intermolecular interactions, heterogeneous methods are widely used when one of the reagents is immobilized on a carrier and the other is in solution. These methods include affinity chromatography,11,12 microarray analysis,13 and recording of binding in flow cells.14,15 However, they are not optimal; the resulting KD value is affected by conformational rearrangements during immobilization and by diffusion limitations of interactions between solved and immobilized reagents. The ability to form an optimal three-dimensional configuration can be critical for the aptamer’s binding activity. Therefore, using homogeneous methods is preferable for determining KD.
Homogeneous methods for measuring constants are divided into two groups. The first group consists of methods based on the separation of the formed complex and unbound components after the reaction in solution. A widely used separation method is equilibrium dialysis.10,16-18 However, it has significant drawbacks: the duration and need for relatively large volumes of reagents.10,19 The second group includes methods in which the formation of the ligand-receptor complex leads to a change in the physical parameter (mobility, fluorescence, heat capacity, etc.). This group includes isothermal titration calorimetry,20,21 microscale thermophoresis,22-24 capillary electrophoresis25-27 and recording the polarization/anisotropy of fluorescence.28-32 All of these approaches have advantages and disadvantages. Isothermal titration calorimetry does not require additional labels, but it is a long-lasting method with a large consumption of reagents. Microscale thermophoresis is characterized by low sample consumption (a few microliters), but it requires expensive equipment.33 The use of capillary electrophoresis requires a significant difference of electrophoretic mobility between initial interacting compounds and their complexes. This technique is restricted when aptamer binds a small target due to the difficulties of aptamer and aptamer-ligand complex separation.34,35 In this paper, we consider the measurement of constants using registration of fluorescence anisotropy (FA). FA is a value reflecting the ability of fluorophores to depolarize absorbed plane-polarized light during fluorescence. The FA value is determined by the mobility of the fluorophore in the
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solution, which depends on the dimensions of the complex into which the fluorophore enters. Therefore, the formation of a complex with a fluorophore-labeled molecule leads to a change of FA. For accurate and sensitive evaluation of a complexation based on FA measurements, the mobility of the labeled molecule and its complex in the solution should be very different. FA (r) is determined by the formula ∥ −
= , (1) ∥ + 2
where I∥ is the intensity of the fluorescence component parallel to the polarization of the exciting light, and I⊥ is the intensity of the fluorescence component perpendicular to the polarization of the exciting light. The FA registration during complexation has several advantages, including no need for sorption of reagents on the substrate and subsequent separation of reacted and unreacted molecules, and short reaction times. In addition, the FA of a mixture of free labeled molecule and its complex with the receptor is additively determined by the ratio of the states36
= ,
(2)
where r is the anisotropy of the mixture, ri is the anisotropy of the i-th component of the mixture, and is the fractional intensities of the i-th component of the mixture. Because of its advantages, FA is of interest for evaluating binding parameters. FA-based methods are used to characterize the antibody-antigen interactions37-39 and screening for low molecular weight enzyme inhibitors.40-42 The specific feature of fluorescence anisotropy as the controlled parameter for interacting molecules is its most pronounced changes (i.e., significant deceleration of rotation) when a small compound with fluorescent properties is included in a big complex. Due to this, the fluorescence anisotropy is a useful parameter to control the binding between low molecular ligands (with typical M.W. 102 Da) and aptamer receptors (M.W. > 104 Da). The FA application for such pairs was described in30,31 and was based on recording changes in the intrinsic FA of the targets (ligands). However, only targets with pronounced fluorescent properties are applicable for such measurements. The key demand for the application of the proposed technique is significant changes of FA after binging fluorescently labeled targets with aptamer. Therefore, it is efficient for low molecular weight targets. In the case of macromolecular targets, the changes in FA decreased, and the possibilities of reliable quantitative measurements are limited. The approaches based on the use of labeled aptamers instead of labeled ligands could be alternatives for this case.28,43 Fluorescent-labeled aptamers also were applied to measure interaction with low molecular targets.44,45 But the principle is not the same as for macromolecular targets. It is based on aptamer conformational change and is induced by a change of FA of the label, which is covalently attached to the aptamer. This approach may be useful, but its efficiency depends significantly on such nonpredictable parameters as conformational-caused changes of fluorescence anisotropy. Hence, the measurement of the dissociation constant by this approach seems to be complicated. The competitive approach is similar to the existing practice of ELISA described by Friquet et al.46 and then widely
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used.47,48 In this practice, the ligand (in native dissolved form) and its derivative (conjugate with protein carrier in immobilized form) compete as antigens for binding with dissolved antibodies, and a special protocol is used to calculate definitively the binding constant for the native ligand. However, the use of such competition for FA-based determination of constants is not practiced for aptamers. The only competitive scheme for the determination of constants using a fluorescently labeled ligand was considered only by Wang et al.49 using aminoglycoside antibiotics; however, this development needs methodological improvement. The authors did not consider changes in label fluorescence after complexation and consequent further distortions in detail. The choice of reagents’ concentrations for experiments was not detailed. Both of these issues are analyzed in this article. We have proposed a general approach for obtaining the dissociation constant of the aptamer-ligand complex based on the registration of FA under competitive interaction of the native ligand (L) and the fluorophore-labeled ligand (L*) with the aptamer. The approach is based on two experimental blocks: (1) Determination of KD1, the dissociation constant of the complex between the aptamer and the ligand labeled with the fluorescein derivative (L*). The KD1 determination is based on the change in FA during the interaction of L* with the aptamer. The approach includes obtaining several FA dependencies from the aptamer concentration at different concentrations of L*. (2) Determination of KD2, the dissociation constant of the complex between the aptamer and the native, unlabeled ligand (L). To determine KD2, FA measurements were carried out in a system with a competition between L* (fixed concentration) and L (variable concentration) for a limited number of aptamer binding sites. Several competitive curves were obtained at different aptamer concentrations. Specific requirements for the implementation of this approach and their justifications are presented in the Results and Discussion section. The study included (1) the choice of requirements for conditions of measurements and data processing, (2) experimental implementation of these requirements, and (3) verification of the results using equilibrium dialysis as a classical verifying method. Ochratoxin A (OTA) and the specific aptamer that binds OTA were selected as the ligand-receptor pair for this study. OTA is a very common toxic food contaminant with a variety of negative effects.50 Its maximum permissible content in food products is regulated by legislation.51,52 The chosen aptamer, obtained by Cruz-Aguado and Penner,18 is the most often used for the development of aptameric systems for OTA detection.31
EXPERIMENTAL SECTION Reagents, materials, and apparatus. The analytical standard of OTA was obtained from Hromresurs (Russia), and the 4'(aminomethyl)fluorescein hydrochloride was obtained from Thermo Fisher Scientific (USA). The DNA oligonucleotide 5′GAT-CGG-GTG-TGG-GTG-GCG-TAA-AGG-GAG-CATCGG-ACA-3′18 was custom-synthesized, and purified by Syntol (Russia). Tris(hydroxymethyl)aminomethane; dimethyl sulfoxide; fluorescein isothiocyanate; N,N'dicyclohexylcarbodiimide; and dimethylformamide were
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Analytical Chemistry
obtained from Sigma-Aldrich (USA). All the chemicals, including compounds of buffer solutions, were of analytical or chemical reagent grade. Based on the previous practice of the work with the given aptamer, the use of 20 mM Tris buffer, pH = 8.5, containing 120 mM NaCl of 5 mM KCl and 20 mM CaCl2 for efficient interaction with the target was chosen.18,30 The efficiency of the obtained final composition was confirmed in our previous paper.53 A Simplicity Milli-Q® water purification system from Millipore (Germany) was used to obtain ultrapure water for the preparation of buffers and reagent solutions. TCL Silica gel 60 F254 plates from Merck (Germany) were applied for thin-layer chromatography. Fluorescence spectra were obtained using RF-6000 spectrofluorophotometer (Shimadzu, Japan) in 2D spectrum mode. Black nonbinding 96-well microtiter plates from Thermo Scientific NUNCtm (Denmark) were used for FA measurements with a Zenyth 3100 multimode plate reader (Anthos Labtec Instruments, Austria) with the following filter settings: excitation of 485 nm, emission of 535 nm, estimation time of 4 s, and G factor of 0.62. Micro-Equilibrium Dialyzers (Harvard Apparatus, UK) were used for studies based on equilibrium dialysis. To measure the fluorescence intensity of OTA in equilibrium dialysis experiments, a 2300 EnSpire microplate reader (PerkinElmer, USA) was used at an excitation wavelength of 375 nm and an emission wavelength of 430 nm. Synthesis of the fluorescein-labeled OTA derivative (OTA-Flu). The carbodiimide protocol with modifications54 was used for the synthesis of fluorescein-labeled OTA; 4 mg of OTA was dissolved in 0.2 mL of dimethylformamide, which also contained 3 mg of N-hydroxysuccinimide and 5 mg of N'-dicyclohexylcarbodiimide, and, after stirring, was incubated overnight at room temperature. Then 5 mg of 4'(aminomethyl)fluorescein was added to the obtained solution. The mixture was incubated in the dark for 2 hours and then separated by thin-layer chromatography with the chloroform:methanol eluent (4:1 volume ratio). The stock OTA-Flu solution was prepared by dissolving the main band (Rf = 0.9) in methanol and was stored at 4 °C. Flu-OTA concentration was determined by a calibration curve with known fluorescein concentrations. The working solutions of Flu-OTA were prepared using the dilution with TB. Fluorescence spectroscopy of OTA, OTA-Flu, and their complexes with aptamer. To obtain the spectrum of aptamerlabeled and native ligand complexes, we mixed 750 µL of OTA (480 nM) or OTA-Flu (12 nM) solution in TB with 750 µL of aptamer (2 µM) solution in TB. To obtain the spectrum of free OTA and OTA-Flu, we used 750 µL of TB instead of the aptamer solution. In addition, we obtained the fluorescence spectrum of 1.5 µL aptamer (1 µM) solution in TB. The spectra were measured in 1 cm quartz cuvettes. The excitation wavelengths were 375 nm for OTA and 485 nm for OTA-Flu, respectively. Interaction of aptamer with labeled ligand. Dilutions of the aptamer in the range from 8 µМ to 2 nM in TB (20 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, and 20 mM CaCl2, pH 8.5) were prepared, and 100 µL of these dilutions and 100 µL of OTA-Flu (concentrations 27.2, 13.6, 6.8, 3.4, 1.7, and 0.9 nM) were added to the wells of a microtiter plate. The plate was softly shaken for 5 min at room temperature, FA values were measured, and dependences of normalized FA (∆ri = ri –
rf, where rf is FA of free OTA-Flu, and ri is FA for the i-th sample) from the aptamer concentration were obtained. Competitive interaction of aptamer with native and labeled ligand’s. Solutions of aptamer (concentrations 4800, 2400, 1200, 800, 600, 480, and 200 nM) and OTA (concentrations ranging from 10 µМ to 0.4 nM) in TB were prepared, and then wells of microtiter plate were consistently filled with 100 µL of the appropriate OTA solution, 50 µL of 12 nM OTA-Flu solution in TB, and 50 µL of aptamer solution. The mixtures TB + OTA-Flu + aptamer and TB + OTA-Flu + TB were used to determine the maximum and the minimum degree of anisotropy, respectively. The FA measurements were then carried out and processed in the same way, as described in Section above. Seven competitive dependences of normalized FA from the OTA concentration were obtained. Equilibrium dialysis binding experiment. Equilibrium dialysis was carried out according to the protocol described by Cruz-Aguado18 and McKeague,31 with minor modifications. Dilutions of the aptamer in the range from 24 µМ to 11 nM in 150 µL of TB (and TB without aptamer as negative) were prepared and mixed at equal volumes with 240 nM OTA solution in TB. Loading chambers of the dialyzers were loaded with 300 µL of the mixtures, and receiving chambers received the same volume of TB. The chambers were separated by acetate nitrocellulose membrane MWCO 10 kDa (Harvard Apparatus, UK). Incubations were performed for 48 h at room temperature. Then 100 µL of aliquots were taken from each chamber, transferred to the wells of a microtiter plate, and mixed with 100 µL of TB. The fluorescence of OTA was then measured.
RESULTS AND DISCUSSION Development of a step by step scheme for the determination of the aptamer-ligand interaction constant. We have developed and demonstrate the requirements for experiments on the aptamer-labeled ligand binding and the competition between the native and labeled ligand for binding to the aptamer, including the mathematical apparatus for calculating the constants, the requirements for excluding the distortion of the results when measuring FA and minimizing errors in measuring constants. The determination of KD1 for the aptamer-labeled ligand interaction. The interaction of the aptamer with OTA labeled with fluorescein occurs in a ratio of 1 to 1 18 and is described by + ∗ ↔ ∗ ,
where [R] is the equilibrium concentration of the aptamer, [L*] is the equilibrium concentration of the labeled ligand, and [RL*] is the equilibrium concentration of their complex. In the experiment, OTA-Flu is used in a constant concentration, and the concentration of the aptamer varies. The reagent ratio is chosen so that the concentration of the labeled ligand is much less than the aptamer concentration ([L*]≪[R]). The duration of the incubation ensures achievement of chemical equilibrium. Under the selected conditions, the dissociation constant is expressed by (∗ − ∗ ) = , (3) ∗
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where [R]T is the initial concentration of the receptor (aptamer), and [L*]T is the initial concentration of the labeled ligand. Because FA is detected in the experiment to calculate the binding constant, it is necessary to express the concentration of reagents via FA. In a system containing free and labeled ligands bound to an aptamer, the fluorescence fraction of the labeled ligand bound to the aptamer is55 − r( f"#$%& = , (4) " − r(
where ri is the FA of the i-th point of the dependence on the aptamer concentration, rf is FA at zero concentration of the aptamer, and rb is the FA limit at the maximum aptamer concentration and the transition of the entire labeled ligand to the bound state. If binding to the aptamer does not change the fluorescence intensity (quantum yield) of the fluorophore, then the portions of mixture components in terms of intensity and concentration are equal. (The change in fluorescence intensity as a result of binding will be considered in Section “Influence of changes in fluorescence on the results of the experiment”.) We transform equation (4), replacing the fluorescence anisotropy with the concentration of reagents: rb is FA when the entire labeled ligand is bound to the aptamer ([RL*]100% = [L*]T); we substitute rb by [L*]T. rf is FA when there is no binding of the labeled ligand to the aptamer ([RL*]0 = 0); we substitute rf by 0. ri is FA corresponding to the i-th concentration of the aptamerligand complex; we substitute ri by [RL*]. We obtain the equation for the concentration fraction of the bound ligand (Fbound), expressed in terms of concentrations as ∗ *+,-./ = ∗ . (5) Substituting in equation (3) the value of [RL*], expressed through Fbound according to equation (5), we obtain the equation connecting the dissociation constant and the registered FA as т = − т . (6) *+,-./ As follows from equation (6), if Fbound = 1/2, then KD1 = [R]T; that is, the 50% binding point (IC50) corresponds to KD1. For the most accurate measurement of KD1, the 50% binding point was determined using a four-parameter sigmoid fitting of the FA dependence on the aptamer concentration as " − r( (7) = r( + , 1 + (4/46 )7 where ri is the FA for the i-th point; x is the concentration of the reagent (in this experiment of the aptamer) plotted along the abscissa axis at the i-th point; rb is the FA for an infinitely high concentration of the reagent (upper asymptote); rf is the FA for an infinitely low concentration of the reagent (lower asymptote); x0 is the concentration of the reagent at the inflection point (the point of interest of 50% binding); and p is the slope of the curve at the inflection point. This experiment not only determines KD1 but also allows us to choose the concentration of OTA-Flu for a competitive experiment (see Section “The influence of receptor and labeled ligand concentrations on the measurement error of the dissociation constant”).
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The determination of KD2 for the aptamer-native ligand interaction. Having determined KD1, we proceed to measure the interaction constant of aptamer-unlabeled ligand (KD2) based on a study of a system containing an aptamer, a labeled ligand, and an unlabeled ligand. In these experiments, OTAFlu and aptamer concentrations are constant, and OTA concentration varies. The duration of the incubation ensures achievement of chemical equilibrium, in which the interactions are described by 9:;
+ ∗
+
