title: Purification of pre-miR-29 by a new O-phospho-l-tyrosine affinity chromatographic strategy optimized using design of experiments.
abstract: MicroRNAs are the most studied small non-coding RNA molecules that are involved in post-transcriptional regulation of target genes. Their role in Alzheimer's disease is being studied and explored in order to develop a new therapeutic strategy based on specific gene silencing. This disease is characterized by protein deposits, mainly deposits of extracellular Aβ plaques, produced upon endoproteolytic cleavage of APP by ß-site APP-cleaving enzyme 1 (BACE1). Recent studies have shown that particularly miR-29 cluster can be involved in the decrease of Aβ plaques production, by acting on BACE1 expression silencing. In order to use this microRNA as potential therapeutic it is essential to guarantee its purity, stability and integrity. Hence, the main purpose of this study was the development of a new affinity chromatographic strategy by using an O-phospho-l-tyrosine matrix and applying Box-Behnken design (BBD) to obtain pre-miR-29 with high purity degree and yield, envisioning its application in gene therapy. Thus, after process optimization the best results were achieved with a decreasing ammonium sulfate gradient in 10mM Tris buffer, pH 8 (1.6M (NH4)2SO4, 1.11M (NH4)2SO4 and 0M (NH4)2SO4), at 16°C. These experimental conditions allowed the recovery of pre-miR-29 with 52% of purity and 71% of recovery yield. The O-phospho-l-tyrosine matrix was initially chosen to mimic the natural interactions that occur inside the cell, and in fact it was proved a satisfactory selectivity for pre-miR-29. Also the innovative application of BBD for this strategy was efficient (R(2)=0.98 for % relative recovery and R(2)=0.93 for % relative purity) and essential to achieve best purification results in short time, saving lab resources.
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: Adriana
authors.initials: A
authors.lastname: Afonso
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Pereira
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: João A
authors.initials: JA
authors.lastname: Queiroz
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: Ângela
authors.initials: Â
authors.lastname: Sousa
authors.affiliation: 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.
authors.forename: Fani
authors.initials: F
authors.lastname: Sousa
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D021621:Imaging, Three-Dimensional; D035683:MicroRNAs / Q000737:chemistry / Q000302:isolation & purification*; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry*
chemical_list: D035683:MicroRNAs; C000591550:O-phospho-L-tyrosine; D014443:Tyrosine
keywords: Affinity chromatography; Box–Behnken design; Design of experiments; l-Tyrosine; pre-miR-29