Towards the Selection of Single‐Stranded DNA Molecular Recognition Element Against Cyanotoxin L‐BMAA

Abstract

Global warming, anthropogenic pressure and eutrophication contribute to the formation and persistence of cyanobacterial blooms in aquatic environments. Cyanotoxins produced during these blooms represent a threat to ecosystems, commercial industries and human health. B‐N‐methylamino‐L‐alanine (L‐BMAA) is a neurotoxic amino acid that has been linked to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC), Alzheimer’s disease (AD) and Amyotrophic Lateral Sclerosis (ALS). Research has shown that L‐BMAA can act as a glutamate receptor antagonist and can incorporate into proteins occasioning protein misfolding that leads to detrimental processes in brain tissues. Currently, the detection of L‐BMAA is costly, time‐consuming and labor‐intensive. Many research groups around the world are working towards the development of bioanalytical methods to improve the detection of these toxins, including the development of molecular biorecognition elements (MREs) that can provide analyte specificity in biosensing applications. Among MREs, aptamers are well‐known to possess an extraordinary target recognition due to the formation of unique three‐dimensional structures capable of binding specifically to their respective targets.We aim to perform an in vitro selection of a single‐stranded DNA (ssDNA) aptamer that can act as an MRE against L‐BMAA, using the systematic evolution of ligands by exponential enrichment (SELEX) technique. We have worked on the optimization of incubation conditions, PCR amplification parameters and the generation of ssDNA using two different ssDNA libraries, with randomized regions of varying lengths. To facilitate the separation of bound and unbound ssDNA after incubation with the target, we modified L‐BMAA with a biotinylated linker to immobilize it in streptavidin‐coated magnetic beads. For each library, we have identified the appropriate annealing temperature, primer concentrations and number of cycles to favor the amplification of the main product while minimizing by‐product formation. The most challenging step of the SELEX procedure has been the generation and purification of ssDNA in sufficient amounts to continue to the next round of selection. To achieve this, we have tried four main methods: exonuclease digestion, size separation derived from uneven primers, asymmetric PCR and alkaline denaturation using sodium hydroxide. Hitherto, we have completed the first round of selection with both libraries, by performing large‐scale PCR reactions and alkaline denaturation followed by ethanol precipitation to generate the ssDNA. We are working in the first round of negative selection to discard those sequences with affinity to the immobilization linker and enrich only those that bind exclusively to L‐BMAA. Given that our target is a small molecule, our goal is to conduct a stringent SELEX process to obtain a highly specific MRE that can be incorporated into an environmental sensing device.