RACE-SEQ and population-wide polymorphism susceptibility testing for endonucleotically active, RNA-targeting therapeutics: RACE-SEQing RNA cleaving drug mechanism of action

Louise Usher, Pantazis Theotokis, Sterghios Moschos

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

10 Downloads (Pure)

Abstract

High throughput sequencing of the products of 5’ RNA ligase-mediated rapid amplification of cDNA ends (5’ RLM-RACE) reactions (RACE-SEQ) enables the mapping and digital enumeration of expected and novel 5’ ends in RNA molecules. The resulting data are essential in documenting the mechanism of action and precision of endonucleotically active, RNA-targeting drugs such as RNase H-active antisense or small interfering RNA. When applied to error-prone replication systems such as RNA viruses or in vitro RNA replicon systems, the method can additionally report the relative susceptibility of known and unknown polymorphisms to a prospective sequence-specific drug, making it a powerful tool in patient selection and stratification, as well as resistance prediction.
We describe the preparation of sequencing libraries for ultra-high depth 5’ RLM-RACE analysis on two popular second-generation high throughput sequencing platforms (Illumina, Ion Torrent) and supply a detailed bioinformatic analysis pipeline for target site activity definition and enumeration. We further illustrate how the pipeline can be simply modified to generate polymorphism-specific drug susceptibility data from in vitro replicon experiments (RACE-SEQ-MM), in a patient-free manner, to cover both known and unknown target site variants in the population.
Original languageEnglish
Title of host publicationOligonucleotide-based Therapies
EditorsOlof Gissberg, Rula Zain, Karin Lundin
Place of PublicationNew York, NY
PublisherSpringer
Pages283-305
ISBN (Electronic)9781493996704
ISBN (Print)9781493996698
Publication statusPublished - 2019

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Number2036
ISSN (Print)1064-3745

Fingerprint Dive into the research topics of 'RACE-SEQ and population-wide polymorphism susceptibility testing for endonucleotically active, RNA-targeting therapeutics: RACE-SEQing RNA cleaving drug mechanism of action'. Together they form a unique fingerprint.

Cite this