In vitro RNA SELEX for the generation of chemically-optimized therapeutic RNA drugs

Kevin T Urak; Sabrina Shore; William M Rockey; Shi-Jie Chen; Anton P McCaffrey; Paloma H Giangrande

doi:10.1016/j.ymeth.2016.03.003

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In vitro RNA SELEX for the generation of chemically-optimized therapeutic RNA drugs

Journal article

Open access

Peer reviewed

In vitro RNA SELEX for the generation of chemically-optimized therapeutic RNA drugs

Kevin T Urak, Sabrina Shore, William M Rockey, Shi-Jie Chen, Anton P McCaffrey and Paloma H Giangrande

Methods (San Diego, Calif.), Vol.103, pp.167-174

07/01/2016

DOI: 10.1016/j.ymeth.2016.03.003

PMCID: PMC4921298

PMID: 26972786

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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.

RNA aptamers

In vitro SELEX

Next-generation sequencing

Modified nucleotides

2′-Fluoro pyrimidines

Recombinant proteins

Illumina sequencing

Mutant T7 RNA polymerase

Details

Title: Subtitle: In vitro RNA SELEX for the generation of chemically-optimized therapeutic RNA drugs
Creators: Kevin T Urak - Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, United States
Sabrina Shore - TriLink BioTechnologies Inc., San Diego, CA 92121, United States
William M Rockey - Department of Radiation Oncology, University of Iowa, Iowa City, IA 52242, United States
Shi-Jie Chen - Department of Physics, Department of Biochemistry, and Informatics Institute, University of Missouri-Columbia, Columbia, MO 65211, United States
Anton P McCaffrey - TriLink BioTechnologies Inc., San Diego, CA 92121, United States
Paloma H Giangrande - Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, United States
Resource Type: Journal article
Publication Details: Methods (San Diego, Calif.), Vol.103, pp.167-174
DOI: 10.1016/j.ymeth.2016.03.003
PMID: 26972786
PMCID: PMC4921298
NLM abbreviation: Methods
ISSN: 1046-2023
eISSN: 1095-9130
Publisher: Elsevier Inc
Grant note: DOI: 10.13039/100000002, name: National Institutes of Health, award: R01CA138503, R21DE019953; DOI: 10.13039/100000983, name: Mary Kay Foundation, award: 9033-12, 001-09; DOI: 10.13039/100001287, name: Elsa U. Pardee Foundation, award: E2766; DOI: 10.13039/100001024, name: Roy J. Carver Charitable Trust, award: RJCCT 01-224
Language: English
Date published: 07/01/2016
Academic Unit: Radiation Oncology; Internal Medicine
Record Identifier: 9984047789302771

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