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.
CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
Patrícia
P
Pereira
CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
Ângela
Â
Sousa
CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
João
J
Queiroz
CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal; CEF-FFUC - Pharmaceutical Studies Center, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.
Ana
A
Figueiras
CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal. Electronic address: fani.sousa@fcsaude.ubi.pt.
Fani
F
Sousa
D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008239:Lysine; D035683:MicroRNAs / Q000302:isolation & purification*; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000302:isolation & purification*; D012329:RNA, Bacterial / Q000302:isolation & purification*; D041903:Rhodovulum; D012685:Sepharose
D035683:MicroRNAs; D012322:RNA Precursors; D012329:RNA, Bacterial; D012685:Sepharose; D008239:Lysine
Affinity chromatography; Lysine-agarose; RNA purification; microRNA; pre-miR-29