title: Determining Functional Aptamer-Protein Interaction by Biolayer Interferometry.
abstract: Short single-stranded nucleic acids called aptamers are widely being explored as recognition molecules of high affinity and specificity for binding a wide range of target molecules, particularly protein targets. In biolayer interferometry (BLI), a simple Dip-and-Read approach in which the aptamer-coated biosensors are dipped into microplate wells is used to study the interactions between an aptamer and its target protein. Here we describe the protocol for the analysis of the interaction between a well-characterized anti-thrombin RNA aptamer with thrombin (Basic Protocol). We also report on the protocol for the affinity screening of a panel of anti-thrombin RNA aptamers with a single phosphorodithioate (PS2) modification, whereby the position of the modification along the RNA backbone is varied systematically (Support Protocol). The PS2 modification uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphodiester backbone linkage. The PS2-modified RNAs are nuclease resistant and several in vitro and in vivo assays have demonstrated their biological activity. For example, combining the PS2 with the 2'-OMe modification affords increased loading of modified small interfering RNA (siRNA) duplexes into the RNA-induced silencing complex (RISC) as well as enhanced gene-silencing antitumor activity. © 2016 by John Wiley & Sons, Inc.
authors.affiliation: Department of Chemistry, Capital Normal University, Beijing, China.
authors.forename: Xinhui
authors.initials: X
authors.lastname: Lou
authors.affiliation: Department of Biochemistry, Vanderbilt University, Nashville, Tennessee.
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
authors.affiliation: AM Biotechnologies, Houston, Texas.
authors.forename: Xianbin
authors.initials: X
authors.lastname: Yang
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D007368:Interferometry / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
chemical_list: D052157:Aptamers, Nucleotide; D011506:Proteins
keywords: affinity; aptamer; biolayer interferometry; nucleic acid-protein interactions; phosphorodithioate