title: Selection strategy to generate aptamer pairs that bind to distinct sites on protein targets.
abstract: Many analytical techniques benefit greatly from the use of affinity reagent pairs, wherein each reagent recognizes a discrete binding site on a target. For example, antibody pairs have been widely used to dramatically increase the specificity of enzyme linked immunosorbent assays (ELISA). Nucleic acid-based aptamers offer many advantageous features relative to protein-based affinity reagents, including well-established chemical synthesis, thermostability, and low production cost. However, the generation of suitable aptamer pairs has posed a significant challenge, and few such pairs have been reported to date. To address this important challenge, we present multivalent aptamer isolation systematic evolution of ligands by exponential enrichment (MAI-SELEX), a technique designed for the efficient selection of aptamer pairs. In contrast to conventional selection methods, our method utilizes two selection modules to generate separate aptamer pools that recognize distinct binding sites on a single target. Using MAI-SELEX, we have isolated two groups of 2'-fluoro-modified RNA aptamers that specifically recognize the αV or β3 subunits of integrin αVβ3. These aptamers exhibit low nanomolar affinities for their targets, with minimal cross-reactivity to other closely related integrin homologues. Moreover, we show that these aptamer pairs do not interfere with each other's binding and effectively detect the target even in complex mixtures such as undiluted serum.
authors.affiliation: Interdepartmental Program in Biomolecular Science and Engineering, University of California Santa Barbara, Santa Barbara, California 93106, USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Gong
authors.forename: Jinpeng
authors.initials: J
authors.lastname: Wang
authors.forename: Kareem M
authors.initials: KM
authors.lastname: Ahmad
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Csordas
authors.forename: Jiehua
authors.initials: J
authors.lastname: Zhou
authors.forename: Jeff
authors.initials: J
authors.lastname: Nie
authors.forename: Ron
authors.initials: R
authors.lastname: Stewart
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
authors.forename: John J
authors.initials: JJ
authors.lastname: Rossi
authors.forename: H Tom
authors.initials: HT
authors.lastname: Soh
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D039302:Integrin alphaVbeta3 / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D052156:SELEX Aptamer Technique / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D052157:Aptamers, Nucleotide; D039302:Integrin alphaVbeta3; D008024:Ligands; D021122:Protein Subunits