Analysis of pre-miR-29b binding conditions to amino acids by using a surface plasmon resonance biosensor

Abstract

The aim of this work was to provide binding information between the recombinant pre-miR-29b and L-arginine/L-lysine by surface plasmon resonance (SPR) and circular dichroism (CD). This information brings important insights concerning the characterization of the microRNA binding onto chromatographic supports using these amino acids as specific ligands. Moreover, it is possible to determine some particular conditions enabling the improvement of the binding specificity of the amino acid ligands used to purify microRNA, preserving their integrity. The binding responses were detectable and reproducible, allowing the determination of the equilibrium dissociation constant (K_D). Considering the binding affinities, it was verified that the pre-miR-29b binds more strongly to L-arginine (K_D between 10⁻⁶ and 10⁻⁷ M) than to L-lysine (K_D between 10⁻⁵ and 10⁻⁷ M). Remarkably, the results revealed that the ligands possess high affinity to RNA molecules using buffers with low salt concentrations and no binding responses were detected with high salt concentrations, suggesting that electrostatic interactions are mainly responsible for the ligand–analyte interaction. Above all, this study showed the importance of SPR for future screening of other ligands that, like the ones described herein, can be used to design novel microRNA purification platforms which will have a significant impact on biopharmaceutical-based therapeutics.