New approach for purification of pre-miR-29 using lysine-affinity chromatography

miRNA-based gene therapy applications require microRNA with high purity degree, good quality and biologically active. Owing to the commercial interest in these approaches, there is a growing interest in the development of innovative procedures to easily and efficiently purify the RNA. Thus, several chromatographic and non-chromatographic methods have been reported to accomplish this purpose, but not all of these strategies allow the efficient separation of miRNAs. The present study describes a new strategy that uses a lysine ligand in affinity chromatography to efficiently separate pre-miR-29 from a small RNAs mixture. The interest on this biomolecule is related to the fact that pre-miR-29 deficiencies or excesses have been associated to a number of clinically important diseases. The retention behaviour of pre-miR-29 was characterized and adjusted to achieve higher specificity in this chromatographic operation, using an ammonium sulfate stepwise gradient. Overall, it was verified that lysine-agarose support can promote a specific interaction with the pre-miR-29 favouring its total separation. The results also suggest that the underlying mechanism involves biorecognition of pre-miR-29 by the lysine ligands, resulting from the occurrence of different elementary interactions, including hydrogen and hydrophobic interactions.