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. 2021 Sep 10;6(37):24209–24217. doi: 10.1021/acsomega.1c03781

Weak Binding of Levamisole by the Cocaine-Binding Aptamer Does Not Interfere with an Aptamer-Based Detection Assay

Aron A Shoara 1, Zachary R Churcher 1, Sladjana Slavkovic 1, Philip E Johnson 1,*
PMCID: PMC8459413  PMID: 34568699

Abstract

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Levamisole is a common and harmful adulterant of street samples of cocaine and can cause electrochemical tests for cocaine to give false negative results. To see if levamisole would interfere with aptamer-based bioassays, we analyzed the binding of levamisole to the cocaine-binding DNA aptamer. At low aptamer concentrations (0.5 to 20 μM) using isothermal titration calorimetry methods and thermal stability measurements, no binding of levamisole to the cocaine-binding aptamer was observed. At higher levamisole concentrations (500 μM), weak binding to the cocaine-binding aptamer was detected using nuclear magnetic resonance (NMR) spectroscopy chemical shift perturbations. NMR-detected titrations show that levamisole binding is competitive with cocaine binding, indicating that both ligands share a common binding site. Finally, we show that the presence of levamisole does not interfere with the photochrome aptamer switch binding assay for cocaine. We conclude that assays using low concentrations of cocaine, and consequently low concentration of levamisole as an adulterant, should be unaffected by the weak binding of levamisole.

Introduction

Aptamers are nucleic acid molecules that bind a target compound. Aptamers exist in nature as a part of a riboswitch1 and can also be selected in vitro to bind a target molecule through a process referred to as SELEX.2−5 These in vitro-selected aptamers are often used in biosensors, as aptamers can be chosen to bind a large range of targets from small molecules to proteins and to cells.6 Despite advances in selecting aptamers, the use of aptamers in different applications is hindered by inconsistent binding properties using different techniques in different sample conditions. These conditions include not just different buffer and salt conditions but often aptamers selected using surface-based methods bind differently when free in solution. This inconsistency is likely true for solution-selected aptamers when used in surface-based assays. A series of studies have reported these binding ability inconsistencies.7−10 One publication suggests using a range of techniques that perform well at different dissociation constant (Kd) values to confirm binding ability.7 While a variety of techniques can measure affinities in the nano- to micromolar range, nuclear magnetic resonance (NMR) spectroscopy is particularly useful in investigating weak binding events in the hundreds of micromolar to millimolar range.11 In this study, we employ a set of different biophysical techniques to analyze the binding of the cocaine-binding aptamer to levamisole across a range of aptamer concentrations, including NMR methods that work well in determining the affinity of weak interactions.

The cocaine-binding aptamer is one of the most well-studied aptamers for several reasons. First, this aptamer changes from being pre-folded in the unbound state to having a ligand-induced folding mechanism as the length of stem 1 becomes shorter.12 For example, MN4 (Figure 1) has six base pairs in stem 1 and is folded both ligand-free and bound. The cocaine-binding aptamer variant MN19 (Figure 1) has three base pairs in stem 1 and is loosely folded or poorly structured when free and becomes structured upon ligand binding.13,14 This structure-switching variant of the aptamer is the version most used in biosensor development as the structure change is exploited to signal binding.

Figure 1.

Figure 1

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Structures of the cocaine-binding aptamers MN4 and MN19 as well as the compounds levamisole and cocaine. Nucleotides in both constructs are numbered in the same manner as in MN4. Dashes between nucleotides indicate Watson–Crick base pairs while dots indicate non-Watson–Crick base pairs.

A second reason the cocaine binding aptamer is so well studied is that it binds an unusually broad range of compounds. Despite being selected to bind cocaine and not the commonly produced cocaine metabolites, the aptamer binds quinine and related quinine-based compounds with much higher affinity than for cocaine.15−17

Levamisole (Figure 1) is a pharmaceutical compound used to treat parasitic worm infections in humans and animals. It is also a commonly used adulterant of cocaine that causes serious debilitating inflammatory complications and skin necrosis.18−20 The presence of levamisole in cocaine also has the effect of interfering with electrochemical-based detection methods for cocaine and giving false negative readings when above a 1:1 molar ratio.21 At present, it is unknown whether levamisole can interfere with aptamer-based assays for cocaine.

In this study, we were interested in determining if levamisole binds the cocaine-binding aptamer and therefore potentially interfering with an assay for cocaine that employs aptamers. We used isothermal titration calorimetry (ITC) and a thermal melt assay to show that at micromolar concentrations of aptamer, levamisole does not bind the cocaine-binding aptamer. We also used NMR spectroscopy at 500 μM DNA aptamer concentration to show that levamisole binds weakly to the cocaine-binding aptamer with an affinity of (0.5 ± 0.1) mM for the MN4 aptamer. With this weak binding affinity, we then showed that levamisole does not interfere with the photochrome aptamer switch assay (PHASA) for cocaine. Other aptamer-based bioassays for cocaine that work in the low micromolar range of cocaine concentration should similarly be unaffected by the presence of levamisole.

Results and Discussion

ITC: Binding of Cocaine to the MN4 Aptamer in the Presence of Levamisole

As an initial gauge to assess if levamisole is bound by the cocaine-binding aptamer, we used ITC methods to measure the affinity of cocaine in the presence and absence of levamisole to the MN4 aptamer. Three ITC experiments were conducted where cocaine and levamisole were titrated into a 20 μM solution of MN4 aptamer solution both separately and as a 1:1 molar ratio mixture (Figure 2). The thermogram in Figure 2a shows that MN4 binds cocaine with the affinity of (4.2 ± 0.3) μM, enthalpy of (−11.8 ± 0.2) kcal/mol, and entropy of (4.6 ± 0.3) kcal/mol. These results are consistent, within error ranges, with previously published values.12,16,22,23 In contrast, the titration of levamisole into the MN4 aptamer does not produce a detectable binding curve, indicating that levamisole is not bound by the MN4 aptamer at these conditions (Figure 2b). In a seperate experiment, cocaine and levamisole were mixed in a 1:1 molar ratio and titrated into the aptamer, binding was observed with an affinity of (4.3 ± 0.3) μM, enthalpy of (−9.4 ± 0.2) kcal/mol, and entropy of (2.2 ± 0.2) kcal/mol (Figure 2c). Together, these results indicate that cocaine binds to the MN4 aptamer equally well in the presence and absence of levamisole.

Figure 2.

Figure 2

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ITC binding thermograms representing the interaction between MN4 and (a) cocaine, (b) levamisole, and (c) 1:1 cocaine/levamisole mixture. The top of each thermogram is raw titration data showing the heat resulting from each injection of ligand into an aptamer solution. The bottom shows the integrated heat plot after correcting for the heat of dilution. Data were acquired at 15 °C in 20 mM Tris (pH 7.4), 140 mM NaCl, 5 mM KCl with MN4 concentration of 20 μM, and ligand concentration of 0.312 mM.

Thermal Stability Assay Using UV Melts

In the photochrome aptamer switch assay (PHASA) for cocaine, employed later in this manuscript, we use the MN19 aptamer covalently labeled with SITS (4-acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid disodium salt; MN19-SITS). To study whether levamisole affects the thermal stability of MN19-SITS, and therefore interfere with the PHASA assay, UV-monitored thermal melt experiments on MN19-SITS in the presence and absence of levamisole were performed. Experiments were run with MN19-SITS at 0.97 μM. We did not observe any significant difference between the thermal melts of MN19-SITS with or without 1 mM levamisole added (Figure 3). The first derivative analyses of these melting plots, while the aptamer is unbound to cocaine, did not show a transition expected for a folded nucleic acid molecule. This indicates that levamisole does not bind and stabilize MN19-SITS in the manner that the binding of cocaine or quinine does.12,24

Figure 3.

Figure 3

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Analysis of the thermal stability of MN19 using UV melting curves. Displayed is the normalized UV absorbance at 260 nm for the MN19-SITS without levamisole (red) and MN19-SITS with 1.1 mM levamisole (blue). Each data point denotes an average of three experiments with the error bars corresponding to one standard deviation. Shown below each absorption plot are the first derivative analyses. Data acquired with 0.97 μM MN19-SITS in 20 mM Tris (pH 7.4), 140 mM NaCl.

Levamisole Binding Detected Using NMR Methods

The binding of levamisole to the cocaine binding aptamer was detected using NMR methods. Levamisole was titrated into a sample of the MN4 aptamer and monitored by recording 1D 1H NMR spectra (Figure 4a). Chemical shift changes of the imino protons as levamisole was added indicate that levamisole is being bound by the MN4 aptamer. Levamisole binds MN4 in fast exchange on the NMR timescale where the population-weighted chemical shift is observed. This indicates relatively weak binding affinity compared to tighter binding ligands such as cocaine and quinine that bind in slow exchange on the NMR timescale.22,25 The positions of the imino proton resonances were tracked as a function of levamisole concentration in order to determine the Kd value by fitting the chemical shift changes to eq 1. The results of these fits are shown in Figure 4b. Four different imino protons were tracked, and their individual Kd values are summarized in Table 1. The average Kd value for MN4 binding levamisole is (0.5 ± 0.1) mM.

Figure 4.

Figure 4

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Levamisole binding by MN4 monitored by one-dimensional 1H NMR. (A) Imino proton region of MN4 titrated with levamisole. Imino resonances marked with colored stars were used to determine the binding affinity. (B) Overlayed plots of the imino protons fit to the equation that describes 1:1 binding of levamisole and MN4. Solid lines represent the fit and the data by the colored symbols. The color of the symbol and fit line match the peaks indicated with the same color in part (A). Experiments were conducted at 5 °C with a 500 μM DNA aptamer sample in 245 mM KCl 5 mM KxHyPO4 pH 6.8 with 10% 2H2O.

Table 1. Kd Values for Levamisole Binding the Cocaine-Binding Aptamers MN4 and MN19 as Determined by NMR-Based Chemical Shift Perturbationa.

  MN4 (pH 6.8) MN4 (pH 6.0) MN4 (pH 8.0) MN19 (pH 6.8)
nucleotide Kd (mM) Kd (mM) Kd (mM) Kd (mM)
T15 0.37 ± 0.06 0.37 ± 0.03    
T18 0.61 ± 0.04 0.37 ± 0.06   0.9 ± 0.2
G29       2.7 ± 0.3
G30 0.48 ± 0.03      
G31 0.51 ± 0.04 0.32 ± 0.04 0.42 ± 0.05 1.7 ± 0.3
T32   0.32 ± 0.05 0.34 ± 0.05  
average 0.5 ± 0.1 0.35 ± 0.03 0.38 ± 0.05 1.7 ± 0.9
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a

The error in the Kd value for individual bases is from the error in fit. The error in the average is the standard deviation of the values used to calculate the average.

Insight into the location of the levamisole binding site is gained by looking at the nucleotides whose chemical shifts move most with ligand binding. These residues are, in order of decreasing magnitude, G31, G30, G4, T18, G29, T15 and G34. With the exception of G4, four of the five nucleotides that change chemical shift most are at, or adjacent to, the three-way junction (Figure 1). This finding matches the location in the MN4 aptamer for cocaine, quinine, and amodiaquine binding.17,22,25 For all cocaine-binding aptamer and ligands studied to date, the nucleotide whose chemical shift changes the most is that of G31. This result indicates that levamisole shares the same binding site as the other ligands for the MN4 aptamer.

To see if levamisole and cocaine compete for the same binding site, the levamisole-bound MN4 sample was then titrated with cocaine. In this titration (Figure S1), by the stage where two molar equivalents of cocaine were added, signals indicative of the cocaine-bound MN4, such the downfield T19 imino, indicate that cocaine is bound, and levamisole is displaced. This indicates that cocaine and levamisole compete at the same site for binding MN4.

We next confirmed the competitive binding cocaine and levamisole by titrating levamisole into a 1:1 molar ratio sample of MN4 bound to cocaine. A sample of MN4 was titrated with cocaine to a 1:1 molar ratio. Then, this sample was titrated with levamisole to see if the levamisole could compete off the bound cocaine (Figure S2). In this experiment, it took ∼50 molar equivalents of levamisole to compete off the 1 equivalent of cocaine from MN4. The competitive binding is best shown by the disappearance of T19 with levamisole binding. This large excess of levamisole is consistent with its weak binding affinity compared with that of cocaine.

To confirm that levamisole binding by MN4 is not simply weak non-specific binding to DNA, we looked at the binding of levamisole to two control sequences for the aptamer. In the first control (MN4-rando), the sequence of MN4 was randomized, but the nucleotide composition was kept constant. For the second control, we used the SS1 sequence where the two AG base pairs (A7/G30 and A21/G29) were switched to be GA base pairs (G7/A30 and G21/A29) as shown in Figure S3. We have previously used SS1 as a negative control for cocaine and quinine binding as it interacts very weakly with these ligands with binding in the 0.5–1 mM range.26 Titrations of levamisole monitored by NMR spectroscopy were performed for MN4-rando and SS1 under the same conditions as for MN4. As seen in Figure S3, the addition of levamisole produced no changes in chemical shift of the imino signals that would indicate levamisole binding. We observe some small changes in peak height that we attributed to a slight dilution of the sample as ligand solution is added. Together, these data show that levamisole does not bind these DNA molecules, indicating that levamisole binding by MN4, though weak, is a result of a specific interaction.

Given the weak binding of levamisole to MN4, we also wished to see if levamisole was bound by the short stem 1 cocaine-binding aptamer variant MN19 that undergoes ligand-induced folding with cocaine binding. A titration of MN19 with levamisole was monitored by 1D 1H NMR spectra and is shown in Figure S4a. Again, changes in chemical shifts of imino signals as levamisole is added indicate that binding is occurring. The positions of the imino proton resonances were tracked as a function of levamisole concentration to determine the Kd value for the interaction by fitting to eq 1 (Figure S4b). The Kd values of three imino signals were averaged to provide an average Kd value for levamisole binding MN19 of (1.7 ± 0.9) mM (Table 1). This weaker ligand binding by MN19 is consistent with MN19 binding weaker to other known cocaine-binding aptamer ligands such as cocaine and quinine.12 This is attributed to some of the binding free energy being used to fold the aptamer, with the remaining binding free energy being detected as an apparent Kd that is weaker than for MN4, an aptamer that does not need to use some of the binding free energy to fold.

We then performed competition binding experiments to see if levamisole and cocaine compete for the same binding site in MN19. First, the MN19-levamisole complex was titrated with cocaine (Figure S5) and signals indicative of the MN19-cocaine complex, such as the downfield T19 and the distinct T28 and T32 signals that are overlapped in the MN19 levamisole complex, were seen by a 2:1 molar ratio of cocaine:levamisole. We also titrated cocaine into MN19 (Figure S6) with the NMR spectrum being identical to what we have seen and assigned previously.13,14,23 This complex was then titrated with levamisole to a final molar ratio of levamisole to cocaine of 50:1 (Figure S6). By a ratio of approximately 30–50:1, the peaks indicative of cocaine-bound MN19 (particularly T19, T32, T28, and G4) were replaced with their levamisole-MN19 counterparts. Together, these experiments show that levamisole and cocaine compete for the same binding site in the MN19 and MN4 aptamer constructs.

The pH dependence of the binding of levamisole by the MN4 aptamer was investigated by repeating the MN4-levamisole NMR titrations in a pH 6 and a pH 8 buffer. We did this as the pKa value of the tertiary amine in levamisole is ∼7, and our initial titrations were performed at pH 6.8 (Figure 4, Figures S1–S6). This could potentially mean approximately half of the levamisole is protonated and half neutral. First, a titration of MN4 with levamisole was conducted in a pH 6 buffer (Figure S7a). At this pH, the imino spectra of free MN4 and levamisole-bound MN4 are essentially identical to the spectra acquired at pH 6.8. To determine the Kd of this interaction, the positions of several imino protons was tracked as a function of levamisole concentration and fit to eq 1 (Figure S7b) with the average Kd value being (0.35 ± 0.03) mM (Table 1). This affinity is very close to being within the error range for the value obtained at pH 6.8.

A titration of MN4 with levamisole in a pH 8 buffer was then performed (Figure S8a). At this pH value, the imino protons of several nucleotides, including T15, G29, G24, and G30 are not observable, likely due to a change in the hydrogen exchange rate at this pH. Binding was still observed, and the imino proton resonance positions of G31 and T32/T28 were tracked as a function of levamisole concentration and fit to eq 1 (Figure S8b). The values of these fits can be seen in Table 1, and the average Kd value at pH 8 is (0.38 ± 0.05) mM. The measured Kd values obtained at pH 8 agree within the uncertainty. Within the pH 6 to pH 8 range tested, there appears to be no appreciable difference in the Kd of the MN4 and levamisole interaction, indicating that binding is not dependent on the protonation state of the tertiary amine or that the pKa value of this atom is significantly different from predicted.

Affinity of MN19-SITS to Cocaine by Fluorescence Anisotropy

We used fluorescence anisotropy to measure the binding affinity of the MN19-SITS aptamer to cocaine in the presence of levamisole. Fluorescence anisotropy correlates to the rotational diffusion of MN19-SITS undergoing Brownian motion of the macromolecule for which the anisotropy is measured. By quantifying the observed polarized fluorescence emitted from SITS upon addition of cocaine, we determined that the affinity of MN19-SITS for cocaine is (19.8 ± 0.9) μM at 15 °C in a complex matrix containing levamisole (Figure 5). The obtained affinity values agree with published Kd values using ITC and intrinsic fluorometry methods.23,26 To validate our method, we measured the concentration limit of detection (CLoD) of cocaine (10 ± 1) μM at 15 °C. The obtained CLoD agrees with our published data.27

Figure 5.

Figure 5

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Fluorescence anisotropy assay for cocaine detection. Shown is the measured fluorescence anisotropy of MN19-SITS as a function of cocaine concentration in a complex matrix containing 150 μM levamisole, 20 mM Tris (pH 7.4), and 140 mM NaCl at 15 °C. The solution is excited at 340 nm and fluorescence anisotropy of MN19-SITS is detected in an emission range of 350–600 nm. Nonlinear regression analysis was used to quantify the apparent Kd of cocaine. The inset displays the linear portion of the plot that is used to calculate the concentration limit of detection (CLoD) of cocaine. Error bars are the experimental standard deviation of five trials.

The Photochrome Aptamer Switch Assay for Cocaine Is Unaffected by Levamisole

We measured the fluorescence kinetics of 0.50 μM MN19-SITS in 20 mM Tris buffer (pH 7.4), 140 mM NaCl at 15 °C, constant excitation of 340 nm, and an emission of 422 nm to detect the effect of levamisole concentration on the photoisomerization of trans-stilbene. We did not detect any significant alterations in the kapp of MN19-SITS versus levamisole (Figure 6). Further analyses of 0.50 μM MN19-SITS as a function of cocaine concentration in a complex matrix that also contains levamisole showed that the apparent rate for the fluorescence kinetics decay (kapp) decreases as the cocaine concentration increases. The linear slope of measured kapp constants of MN19-SITS as a function of cocaine concentration in a complex matrix including levamisole agrees with that in the same complex matrix without levamisole. For a negative control experiment, unconjugated stilbene fluorescence was examined to evaluate non-specific interactions of levamisole in both Tris buffer and the complex matrix (Figure S9). None of the examined conditions exhibited any significant differences in the presence or absence of levamisole.

Figure 6.

Figure 6

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Photochrome aptamer switch assay (PHASA) applied to detect cocaine in a mixture containing levamisole. Shown on the top panel is the fluorescence decay kinetics curves of MN19-SITS titration with cocaine in a complex matrix containing 150 μM levamisole, 20 mM Tris (pH 7.4), and 140 mM NaCl at 15 °C. The gray curve represents the negative control (150 μM levamisole in complex matrix without cocaine). The bottom panel displays normalized linear plots of observed kapp values as a function of the ligand to MN19-SITS molar ratio. The aptamer-SITS conjugate is continuously excited at 340 nm, and emissions at 422 nm are simultaneously detected as a function of time in milliseconds. Red triangles represent cocaine titration in MN19-SITS in a complex matrix containing levamisole; blue squares denote cocaine titration in MN19-SITS and Tris buffer; and black circles signify levamisole titration in MN19-SITS and complex matrix.

Conclusions

In this manuscript, we investigated whether or not levamisole binds the cocaine-binding aptamers MN4 and MN19. Our interest in this comes from the fact that levamisole is a common adulterant of cocaine samples, that levamisole consumption is harmful to the user of an adulterated sample, and reports that levamisole can interfere with electrochemical detection methods. We observe weak binding of levamisole to both MN4 and MN19 (Table 1) resulting from changes in the imino 1H signals in the aptamer (Figure 4). Binding was not observed using ITC methods (Figure 2) or from the detection of a thermal unfolding transition in MN19 (Figure 3) as these two methods work at a much lower concentration in NMR spectroscopy in the micromolar range as opposed to hundreds of micromolars for NMR. As many bioassays work in the micromolar concentration range, we do not expect this weak binding of levamisole by the cocaine-binding aptamer to interfere with aptamer-based assays. We demonstrate this non-interference by showing that the photochrome aptamer switch assay using SITS-conjugated to MN19 is not affected by the presence of levamisole.

Methods

Materials

The cocaine-binding aptamer constructs (Figure 1) were obtained from Integrated DNA Technologies (IDT, Coralville, Iowa) and used without further purification. DNA samples were dissolved in distilled, deionized Milli-Q Ultrapure water with a resistivity of 18.2 MΩ·cm at 25 °C (ddH2O). Then, DNA samples were exchanged three times in 3 kDa molecular weight cut-off concentrators with 1 M NaCl and washed at least three times with ddH2O using centrifugation at 25 °C. Aptamer samples were then exchanged into the buffer of choice for a given experiment. Prior to use, samples were heated in a 90 °C water bath for 3 min then cooled in an ice-water bath for 5 min to favor intramolecular folding of the aptamer.

Cocaine hydrochloride was obtained from Toronto Research Chemicals (Toronto, ON, Canada). SITS (4-acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid disodium salt), levamisole (L[−]-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b] thiazole hydrochloride), and other reagents used were obtained from Sigma Aldrich. All the SITS powder and solutions were kept under dark conditions to prevent random photoisomerization of trans-SITS. To covalently attach SITS to the MN19 aptamer in the PHASA experiments, the MN19 DNA was synthesized with a C6-NH2 group at the 5′ end. Aptamer and ligand concentrations were determined by UV–vis absorbance spectroscopy using calculated extinction coefficients supplied by the manufacturers.

Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) was performed using a MicroCal VP-ITC instrument in a manner similar as previously described.28 The aptamer samples used for the ITC experiments were exchanged into a buffer of 20 mM Tris (pH 7.4), 140 mM NaCl, 5 mM KCl, and stock solutions of the ligands were prepared by dissolving the calculated weight into the binding buffer. Samples were degassed before analysis with a MicroCal Thermo Vac unit. All experiments were corrected for the heat of dilution of the titrant. Titrations were performed with aptamer samples in the cell and with the ligand, the titrant, in the needle.

All ITC binding experiments were performed at 15 °C with an aptamer concentration of 20 μM and using a ligand concentration of 0.312 mM and consisted of an initial delay of 60 s, a first injection of 2 μL, and a 300 s delay. The subsequent 34 injections were 8 μL, spaced every 300 s. The first point was removed from all data sets due to the different injection volume and delay parameters. To obtain the dissociation constant (Kd) and thermodynamic parameters, the ITC data were fit to a one-site binding model using the manufacturer-provided Origin 7 software.

NMR Spectroscopy

NMR experiments were conducted on a 600 MHz Bruker Avance spectrophotometer at 5 °C in a manner described previously.29 Spectra were acquired in 245 mM KCl, 5 mM KHPO4, pH 6.8 in 10% 2H2O, and 90% H2O. NMR data were processed using TopSpin 3.7 (Bruker).

For both MN4 and MN19 aptamers, two samples were prepared from a common stock. Samples started at a concentration of 500 μM and a volume of 550 μL. In one sample, levamisole was titrated into the free aptamer until a molar ratio of 1:10 (aptamer:levamisole) was achieved. After this, cocaine was titrated into the sample to a final molar ratio of 1:10:2 for the MN4 (aptamer:levamisole:cocaine) sample and 1:10:3 for the MN19 (aptamer:levamisole:cocaine) sample. For the second sample for each aptamer, cocaine was titrated to a 1:1 molar ratio (aptamer:cocaine). Levamisole was then titrated into the cocaine-bound samples to a final ratio of 1:1:50 (aptamer:cocaine:levamisole).

To check the effect of pH on the interaction between levamisole and MN4, a sample of MN4 was split in half and exchanged into (i) a pH 6 buffer (245 mM KCl, 5 mM KHPO4, pH 6.0 in 10% 2H2O/90% H2O) and (ii) a pH 8 buffer (200 mM NaCl, 20 mM deuterated ammonium acetate, pH 8.0 in 10% 2H2O/90% H2O). Each of these samples was titrated with levamisole to a final molar ratio of 1:10 (aptamer:levamisole).

The affinity of the aptamer–levamisole interaction was determined using standard methods of following the chemical shift perturbations of signals in the NMR spectrum.30 The fraction bound (fb) was fit as a function of ligand concentration to the equation below:

graphic file with name ao1c03781_m001.jpg 1

where Bmax is theoretical maximum response, Kd is the dissociation constant, and [L] is the concentration of levamisole present in the sample. The fraction bound at a given point was calculated using eq 2:

graphic file with name ao1c03781_m002.jpg 2

where fbi is the fraction bound at a particular point during the titration, δi is the current 1H position of a particular resonance, δ0 is the initial 1H position of the resonance in the absence of any ligand, and δf is the final 1H position of the resonance being used. fbi should range from 0 in the free sample to 1 in the 1:10 aptamer:levamisole sample. This calculation also assumes that the aptamer is fully bound with an fbi of 1 at the final titration point. For MN19, the same scheme as MN4 was used, though peak intensity was used since many peaks in free MN19 are not visible due to the dynamic nature of the unbound MN19 aptamer. Nonlinear curve fits were performed using SigmaPlot 11.

Aptamer-SITS Conjugation

The covalent conjugation of MN19 to trans-SITS was performed through the amine isothiocyanate reaction.27,31 Then, aptamer samples were allowed to equilibrate for 30 min at 20 °C prior to the conjugation reaction. The MN19 aptamer was mixed in 100 molar fold of excess SITS in 100 mM potassium bicarbonate buffer (pH 10) at 150 rpm shaking for at least 12 h under a dark condition to form the MN19-SITS aptamer. The conjugation reactions were done at (20 ± 0.5) °C to retain the secondary structure, as this temperature is below the previously examined thermal denaturation point (Tm) of the MN19 aptamer.24 The MN19-SITS conjugation product was washed and concentrated 10 times using the described centrifugal method to remove unconjugated SITS. To confirm the 1:1 molar ratio of aptamer-SITS products, the relative UV absorbance at 260 nm and 340 nm of aptamer to SITS were measured.

UV melts

UV thermal melt experiments of MN19-SITS in presence and absence of levamisole were performed using a Cary 100 spectrometer and 10-mm fused quartz cuvettes. The temperature increase rate was set at 1 °C/min and controlled by a Cary Peltier unit, and two data points per minute were collected. The DNA melting curves were acquired in a temperature range from 10 to 80 °C. Experiments were performed in triplicate in a complex mixture described previously containing 20 mM Tris buffer, pH 7.4, 140 mM NaCl, and 1.1 mM levamisole.27 For each levamisole–aptamer mixture, a concentration of 0.97 μM the MN19-SITS was chosen to yield ∼0.8 absorbance arbitrary units (a.u.) at 260 nm. The levamisole concentration was kept at ∼1000-fold of the conjugated aptamer concentration. To quantify the thermal shift points (Tm), the first derivative of each thermal curve was plotted as a function of temperature using OriginPro 2016 software.

Fluorescence Anisotropy

We utilized the structure-switching binding mechanism of MN19-SITS in a fluorescence anisotropy assay of MN19-SITS as a function of cocaine concentration in the previously described complex mixture at 15 °C. The fluorescence anisotropy of SITS is governed by the ratio of the polarized light, and it is independent of the absolute emission intensity magnitudes32 Using a pair of manual Cary Eclipse light polarizers, the instrument grating factor (G) was measured as:

graphic file with name ao1c03781_m003.jpg 3

where IHV is the integrated vertical emission intensity of SITS from 350 to 600 nm at the horizontal excitation at 340 nm. Also, IHH is the integrated horizontal emission intensity of SITS from 350 to 600 nm to include the maximum emission peak at 422 nm at the horizontal excitation at 340 nm. Then, the polarized IVV and IVH at each titration point was measured as a function of cocaine concentration, and the fluorescence anisotropy (r) values calculated as stated in eq 4:

graphic file with name ao1c03781_m004.jpg 4

The results from five trials were averaged and plotted as a function of cocaine concentration. To quantify the Kd values and compare the affinities obtained by this method with previously published data, the binding curves were fitted to a one-site binding function and analyzed using the OriginPro 2016 software package.26

Photochrome Aptamer Switch Assay

The fluorescence decay kinetics of MN19-SITS in the absence and presence of multiple levamisole concentrations were examined in 20 mM Tris buffer (pH 7.4) and 140 mM NaCl at 15 °C using a Cary Eclipse fluorescence spectrophotometer and 10 mm fused quartz cuvettes. Then, the fluorescence decay kinetics of MN19-SITS analyzed titrations of cocaine solutions in a complex matrix at 15 °C. The complex matrix conditions included 20 mM Tris buffer (pH 7.4), 140 mM NaCl, and 50 μM each of ATP, atropine sulphate, benzoic acid, choline, creatine phosphate, CTP, dapsone, glycine, GTP, isopropyl β-d-1-thiogalactopyranoside (IPTG), l-ascorbic acid, l-glutamic acid, triethylamine, and UTP as well as 5.5 mM d-glucose, 140 mM glycerol, and 150 μM levamisole. The temperature was kept constant during each trial using a Cary Peltier controller. The spectrofluorometer was optimized for the limit of detection to maintain a constant signal-to-noise ratio, photomultiplier tube voltage, and spectral bandwidth parameters. Maximum UV–vis absorbance and steady-state fluorescence emission wavelengths of levamisole were tested in 20 mM Tris buffer (pH 7.4) and 140 mM NaCl at 15 °C for the inner-filter effect measurements. Since the studied DNA aptamer, cocaine, or levamisole had no light absorbance at (340 ± 10) nm, the inner-filter effects for the loss of the excitation and emission light intensities were not calculated.33

The threshold of detection for the MN19 aptamer conjugated with SITS and the CLoD for cocaine binding were determined from the residual standard deviation obtained from the least square regression of the linear region of the dose–response curve in the presence of 150 μM levamisole.34 We analyzed the CLoD of cocaine using the linear portion of the fluorescence anisotropy and fluorescence kinetic decay of 500 nM aptamer-SITS with varying cocaine concentrations in a complex matrix at 15 °C. The acquired fluorescence kinetics decay constants (kapp) were analyzed as a function of cocaine concentration. The CLoD of aptamer-SITS and the CLoD of cocaine were separately quantified as follows:

graphic file with name ao1c03781_m005.jpg 5

where Sy/x is the calculated standard deviation of the linear regression residuals and m is the fitted linear slope analyzed in the OriginPro 2016 software package.34 To examine the accuracy of the assay in the presence of levamisole, the fluorescence decay kinetics of MN19-SITS were performed in a complex matrix mixed with 150 μM levamisole and previously published values without levamisole at 15 °C. To evaluate the precision of this analysis, relative standard deviation (%RSD) of the mean standard values, or coefficient of variation (%CV), was analyzed as:

graphic file with name ao1c03781_m006.jpg 6

The obtained CV values were then compared with the among-laboratory relative standard deviation (RSDR) exploiting the Horwitz equation:

graphic file with name ao1c03781_m007.jpg 7

The linearity of results was assessed applying a linear regression analysis of normalized average kapp values against cocaine concentration. Also, the range of linear threshold of detection was evaluated in a dose–response curve of fluorescence intensity of trans-SITS as a function of MN19-SITS concentration in a complex matrix containing 150 μM levamisole.35

Acknowledgments

We thank Logan Donaldson (Biology, York University) for the use of his fluorimeter. We also thank Anne Johnson (Ryerson University) and members of the Johnson lab for useful discussions. This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada to P.E.J.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03781.

  • NMR spectra of titrations of cocaine to a levamisole·MN4 complex, levamisole to a cocaine·MN4 complex, levamisole to two control DNA sequences (randomized MN4 and SS1), levamisole to MN19, cocaine to a levamisole·MN19 complex, levamisole to a cocaine·MN19 complex, levamisole to MN4 at pH 6, and levamisole to MN4 at pH 8 and sensing threshold for the photochrome aptamer switch assay in a complex matrix including levamisole (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao1c03781_si_001.pdf (4.8MB, pdf)

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Supplementary Materials

ao1c03781_si_001.pdf (4.8MB, pdf)

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