title: Highly Multiplexed RNA Aptamer Selection using a Microplate-based Microcolumn Device.
abstract: We describe a multiplexed RNA aptamer selection to 19 different targets simultaneously using a microcolumn-based device, MEDUSA (Microplate-based Enrichment Device Used for the Selection of Aptamers), as well as a modified selection process, that significantly reduce the time and reagents needed for selections. We exploited MEDUSA's reconfigurable design between parallel and serially-connected microcolumns to enable the use of just 2 aliquots of starting library, and its 96-well microplate compatibility to enable the continued use of high-throughput techniques in downstream processes. Our modified selection protocol allowed us to perform the equivalent of a 10-cycle selection in the time it takes for 4 traditional selection cycles. Several aptamers were discovered with nanomolar dissociation constants. Furthermore, aptamers were identified that not only bound with high affinity, but also acted as inhibitors to significantly reduce the activity of their target protein, mouse decapping exoribonuclease (DXO). The aptamers resisted DXO's exoribonuclease activity, and in studies monitoring DXO's degradation of a 30-nucleotide substrate, less than 1 μM of aptamer demonstrated significant inhibition of DXO activity. This aptamer selection method using MEDUSA helps to overcome some of the major challenges with traditional aptamer selections, and provides a platform for high-throughput selections that lends itself to process automation.
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Reinholt
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Abdullah
authors.initials: A
authors.lastname: Ozer
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
authors.forename: John T
authors.initials: JT
authors.lastname: Lis
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Harold G
authors.initials: HG
authors.lastname: Craighead
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D015203:Reproducibility of Results; D052156:SELEX Aptamer Technique / Q000295:instrumentation / Q000379:methods*
chemical_list: D052157:Aptamers, Nucleotide