Dr. Séverine Padiolleau from France led a project, supported by ISIDORe, aimed at developing new diagnostic probes for Lyme borreliosis, a tick-borne disease caused by Borrelia burgdorferi. Lyme disease diagnosis has long been the subject of debate due to its complexity, and this project seeks to improve diagnostic tools by creating aptamers—short oligonucleotides that can specifically bind to bacterial surface proteins.
The research team applied SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology, conducting 12 rounds of selection against the bacterial surface protein BbCRASP-2, a key protein on the surface of Borrelia burgdorferi. By using alternating positive and negative selection rounds, and applying selection pressure at certain stages, they aimed to isolate oligonucleotides with high specificity and affinity for the target. Next-generation sequencing (NGS) was used to monitor the progress, which led to the identification of 14 promising aptamer candidates. The project’s goal was to characterize the interaction properties of these aptamers with BbCRASP-2, and this was carried out using BiLayer Interferometry (BLI) at the ROBOTEIN platform.
The results of the study were promising. Six of the aptamer candidates exhibited strong binding affinity, with dissociation constants (KD) lower than 100 nanomolars, while the other eight had higher KD values. Two controls were performed to validate these findings: a control aptamer with a different nucleotide sequence showed weak binding, and tests using an irrelevant target protein confirmed the specific interaction with BbCRASP-2. The six best aptamers were further analyzed using RNAfold, a tool for predicting secondary structures, and the team found that all six contained G-quadruplexes, a structural motif that warrants further investigation.
Looking ahead, the project will extend its analysis using surface plasmon resonance (SPR) to confirm interactions using a different technique. They will also conduct fluorescence-based experiments to ensure that the aptamers can bind to bacterial surface proteins in their natural cellular environment. In addition, the structural conformations of the best aptamers will be examined to improve their effectiveness as diagnostic tools, and their performance in more complex biological samples, such as body fluids, will be tested.