title: Bio-functional surfaces for the immunocapture of AGO2-bound microRNAs.
abstract: MicroRNAs (miRNAs) are endogenous, small (18-24nt), non-coding RNAs that regulate gene expression. Among miRNAs, those bound to the AGO2 protein are the functionally active fraction which mediates the cell regulatory processes and regulate messages exchanged by cells. Several methods have been developed to purify this fraction of microRNAs, such as immunoprecipitation and immunoprecipitation-derived techniques. However, all these techniques are generally recognized as technically complicated and time consuming. Here, a new bio-functional surface for the specific capture of AGO2-bound microRNAs is proposed. Starting from a silicon oxide surface, a protein A layer was covalently bound via epoxy chemistry to orient specific anti-AGO2 antibodies on the surface. The anti-AGO2 antibodies captured the AGO2 protein present in cell lysate and in human plasma. The AGO2-bound microRNAs were then released by enzymatic digestion and detected via RT-qPCR. Control surfaces were also prepared and tested. Every step in the preparation of the bio-functional surfaces was fully characterized from the chemical, morphological and functional point of view. The resulting bio-functional surface is able to specifically capture the AGO2-bound miRNAs from biologically-relevant samples, such as cell lysate and human plasma. These samples contain different proportions of AGO2-bound microRNAs, as reliably detected with the immunocapture method here proposed. This work opens new perspectives for a simple and faster method to isolate not only AGO2-bound microRNAs, but also the multiprotein complex containing AGO2 and miRNAs.
authors.affiliation: Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo (Trento), Italy.
authors.forename: V
authors.initials: V
authors.lastname: Vaghi
authors.affiliation: Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo (Trento), Italy; CNR - Consiglio Nazionale delle Ricerche, Istituto di Biofisica, via alla Cascata 56/C, I-38123 Povo (Trento), Italy. Electronic address: cpotrich@fbk.eu.
authors.forename: C
authors.initials: C
authors.lastname: Potrich
authors.affiliation: Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo (Trento), Italy; CNR - Consiglio Nazionale delle Ricerche, Istituto di Biofisica, via alla Cascata 56/C, I-38123 Povo (Trento), Italy.
authors.forename: L
authors.initials: L
authors.lastname: Lunelli
authors.affiliation: CNR - Consiglio Nazionale delle Ricerche, Istituto di Biofisica, via De Marini 6, I-16149 Genova, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Facci
authors.affiliation: Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo (Trento), Italy.
authors.forename: L
authors.initials: L
authors.lastname: Pasquardini
authors.affiliation: Fondazione Bruno Kessler, Center for Materials and Microsystems, MNF-MateC, via Sommarive 18, I-38123 Povo (Trento), Italy.
authors.forename: L
authors.initials: L
authors.lastname: Vanzetti
authors.affiliation: Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo (Trento), Italy.
authors.forename: C
authors.initials: C
authors.lastname: Pederzolli
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D060565:Argonaute Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D010949:Plasma / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012822:Silicon Dioxide / Q000737:chemistry / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties
chemical_list: C466422:AGO2 protein, human; D000911:Antibodies, Monoclonal; D060565:Argonaute Proteins; D035683:MicroRNAs; D013205:Staphylococcal Protein A; D012822:Silicon Dioxide
keywords: AGO2 immunocapture; Bio-functional surface; MicroRNA purification; MicroRNAs; Surface analysis