Selection, characterization, and biosensing applications of DNA aptamers targeting cyanotoxin BMAA

Abstract

Scientists have established a connection between environmental exposure to toxins like β-N-methylamino-l-alanine (BMAA) and a heightened risk of neurodegenerative disorders. BMAA is a byproduct from certain strains of cyanobacteria that are present in ecosystems worldwide and is renowned for its bioaccumulation and biomagnification in seafood. The sensitivity, selectivity, and reproducibility of the current analytical techniques are insufficient to support efforts regarding food safety and environment monitoring adequately. This work outlines the in vitro selection of BMAA-specific DNA aptamers via the systematic evolution of ligands through exponential enrichment (SELEX). Screening and characterization of the full-length aptamers was achieved using the SYBR Green (SG) fluorescence displacement assay. Aptamers BMAA_159 and BMAA_165 showed the highest binding affinities, with dissociation constants (K_d) of 2.2 ± 0.1 μM and 0.32 ± 0.02 μM, respectively. After truncation, the binding affinity was confirmed using a BMAA-conjugated fluorescence assay. The K_d values for BMAA_159_min and BMAA_165_min were 6 ± 1 μM and 0.63 ± 0.02 μM, respectively. Alterations in the amino proton region studied using solution nuclear magnetic resonance (NMR) provided further evidence of aptamer-target binding. Additionally, circular dichroism (CD) spectroscopy revealed that BMAA_165_min forms hybrid G-quadruplex (G4) structures. Finally, BMAA_165_min was used in the development of an electrochemical aptamer-based (EAB) sensor that accomplished sensitive and selective detection of BMAA with a limit of detection (LOD) of 1.13 ± 0.02 pM.

Fig. 1. Schematic representation of the in vitro selection using Bead-SELEX. During positive selection rounds, BMAA (green spheres) was incubated with the library pool. For the negative selection, the immobilization matrix with no BMAA (gray spheres) was incubated with the library pool. The counter selection involved the incubation of the library with structurally similar compounds (yellow star, orange diamond).

Fig. 2. Schematic representation of the EAB sensor fabrication. Aptamer BMAA_165_min was immobilized on Au electrodes through SAMs. Then, MB was covalently linked to the aptamers via chemical cross-linking, and the electrode surface was blocked with MCH. Electrochemical detection of BMAA was measured using SWV and EIS.

Fig. 3. Sequencing analysis. (A) Base distribution analysis of the relative frequency (%) of each nucleotide across different rounds of SELEX compared to the library control (R0). (B) Abundance (RPM) of the motifs predicted by AptaTRACE (GAGGGG, GGAGGG, GGGAG, GGGGG) and the random sequences chosen for comparison (AAAAA, CCCCC, CCTAGT, TCGATC). (C) Enrichment values of the motifs defined as the abundance in R18 divided by the abundance in R0.

Fig. 4. Affinity evaluation and characterization of aptamers. Evaluation of the binding affinity of full-length aptamers using the SG fluorescence displacement. (A) Binding isotherms of BMAA_159 (K_d = 2.2 ± 0.1), BMAA_165 (K_d = 0.32 ± 0.02) and BMAA_165_scrambled (K_d = no binding). (B) Comparison of the fluorescence response of BMAA and structurally related compounds (AEG, DAB, atenolol). A full analysis of the independent experiments can be found in the ESI (Fig. S7, Table S6†). Evaluation of the binding affinity of truncated aptamers using BMAA-conjugated fluorescence assay. (C) Binding isotherms of BMAA_159_min (K_d = 6 ± 1) and BMAA_165_min (K_d = 0.63 ± 0.02). A full analysis of the independent experiments can be found in the ESI (Fig. S8, Table S7†). Measurements were performed in triplicates and the error bars represent the calculated standard error. The K_d values were determined through non-linear regression analysis by fitting the data with one site-specific binding equation using GraphPad Prism 10.2.0. Qualitative affinity evaluation of the truncated aptamers using the frequency intensity changes at the amino proton region measured with solution NMR. (D) Binding isotherms of BMAA_159_min and BMAA_165_min. The amino proton region of the ¹H NMR spectra is provided in the ESI (Fig. S6†). Spectrums are the average of 2048 transients and error bars represent the signal/noise figure for each measurement. CD spectra of BMAA (10 μM), aptamers (5 μM), and aptamers incubated with BMAA (1 : 2 ratio) recorded in buffer (100 mM KCl, 5 mM MgCl_2, and 20 mM Tris, pH of 7.6) for (E) BMAA_159_min and (F) BMAA_165_min. Each CD spectra is the average of 5 scans.

Fig. 5. Electrochemical detection of BMAA. Characterization of Au electrodes before and after aptamer modification carried out in 5 mM [Fe(CN)₆]^3−/4− redox couple solution (A) CV and (B) Nyquist plot. Electrochemical detection of the EAB sensor upon addition of varying concentrations of BMAA (0, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 pM) performed in buffer solution (4 mM NaCl, 0.2 mM MgCl₂, and 0.8 mM Tris at pH 7.4). (C) Changes in current measured with SWV. (D) Analytical response using the absolute value of Z_R (Ω) obtained with EIS. (E) Calibration curve of the analytical response |Z_R| (Ω) against BMAA concentration (Y = 90.77x + 39, R = 0.999, LOD = 1.13 ± 0.03, LOQ = 1.46 ± 0.07). Measurements represent the mean of three independent experiments (n = 9) and the error bars represent the calculated standard error. A full analysis of the independent experiments can be found in the ESI (Fig. S9 and S10, Table S8†). (F) Comparison of the analytical response |Z_R| (Ω) of BMAA and structurally related compounds (AEG, DAB, atenolol). Measurements were performed in triplicates and the error bars represent the calculated standard error. Experiments can be found in the ESI (Fig. S11†).