title: In vitro RNA SELEX for the generation of chemically-optimized therapeutic RNA drugs.
abstract: Aptamers are single-stranded DNA or RNA oligonucleotides that can bind with exquisitely high affinity and specificity to target molecules and are thus often referred to as 'nucleic acid' antibodies. Oligonucleotide aptamers are derived through a process of directed chemical evolution called SELEX (Systematic Evolution of Ligands by Exponential enrichment). This chemical equivalent of Darwinian evolution was first described in 1990 by Tuerk & Gold and Ellington & Szostak and has since yielded aptamers for a wide-range of applications, including biosensor technologies, in vitro diagnostics, biomarker discovery, and therapeutics. Since the inception of the original SELEX method, numerous modifications to the protocol have been described to fit the choice of target, specific conditions or applications. Technologies such as high-throughput sequencing methods and microfluidics have also been adapted for SELEX. In this chapter, we outline key steps in the SELEX process for enabling the rapid identification of RNA aptamers for in vivo applications. Specifically, we provide a detailed protocol for the selection of chemically-optimized RNA aptamers using the original in vitro SELEX methodology. In addition, methods for performing next-generation sequencing of the RNAs from each round of selection, based on Illumina sequencing technology, are discussed.
authors.affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, United States.
authors.forename: Kevin T
authors.initials: KT
authors.lastname: Urak
authors.affiliation: TriLink BioTechnologies Inc., San Diego, CA 92121, United States.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Shore
authors.affiliation: Department of Radiation Oncology, University of Iowa, Iowa City, IA 52242, United States.
authors.forename: William M
authors.initials: WM
authors.lastname: Rockey
authors.affiliation: Department of Physics, Department of Biochemistry, and Informatics Institute, University of Missouri-Columbia, Columbia, MO 65211, United States.
authors.forename: Shi-Jie
authors.initials: SJ
authors.lastname: Chen
authors.affiliation: TriLink BioTechnologies Inc., San Diego, CA 92121, United States.
authors.forename: Anton P
authors.initials: AP
authors.lastname: McCaffrey
authors.affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, United States; Department of Radiation Oncology, University of Iowa, Iowa City, IA 52242, United States. Electronic address: paloma-giangrande@uiowa.edu.
authors.forename: Paloma H
authors.initials: PH
authors.lastname: Giangrande
mesh_terms: D052157:Aptamers, Nucleotide / Q000138:chemical synthesis* / Q000302:isolation & purification; D001483:Base Sequence; D015195:Drug Design; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D052156:SELEX Aptamer Technique; D017423:Sequence Analysis, RNA
chemical_list: D052157:Aptamers, Nucleotide
keywords: 2′-Fluoro pyrimidines; Illumina sequencing; In vitro SELEX; Modified nucleotides; Mutant T7 RNA polymerase; Next-generation sequencing; RNA aptamers; Recombinant proteins