Profiling the Mismatch Tolerance of Argonaute 2 through Deep Sequencing of Sliced Polymorphic Viral RNAs

Theotokis, Pantazis, Usher, Louise, Kortschak, Christopher K., Schwalbe, Ed and Moschos, Sterghios (2017) Profiling the Mismatch Tolerance of Argonaute 2 through Deep Sequencing of Sliced Polymorphic Viral RNAs. Molecular Therapy - Nucleic Acids. ISSN 2162-2531

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Low allelic and clonal variability among endogenous RNA interference (RNAi) targets has focused mismatch tolerance studies to RNAi-active guide strands. However, the inherent genomic instability of RNA viruses such as hepatitis C virus (HCV) gives rise to quasi-species mutants within discrete clones: this facilitates mismatch tolerance studies from a target perspective. We recently quantified the slicing imprecision of Argonaute 2 using small interfering RNA (siRNA) analogues of the DNA-directed RNAi drug TT-034 and next generation sequencing of 5’ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RACE-SEQ). Here, we present an open source, customizable, and computationally light RACE-SEQ bioinformatic pipeline, describing adaptations that semi-quantitatively report the impact of RNAi hybridisation site mismatches from the target perspective. The analysis shows Argonaute 2 has a substitution-specific, 3-5 log activity window between fully complementary targets and targets with mismatches across positions 10-11. It further focuses the endonucleotic Slicer imprecision around positions 13-17, demonstrating its dependence on guide strand central region complementarity, and potentiation by even a single mismatch. We further propose pharmacogenomics value in testing endogenous targets using recombinant replicon systems and RACE-SEQ to report the pharmacodynamics of sequence-specific oligonucleotide therapeutics against all possible polymorphisms in a population, in a minimally-biased, patient-free manner.

Item Type: Article
Uncontrolled Keywords: siRNA, antisense, oligonucleotide therapeutics, mechanism of action, assay, companion diagnostics, next generation sequencing, precision medicine
Subjects: A100 Pre-clinical Medicine
A300 Clinical Medicine
C100 Biology
C400 Genetics
C500 Microbiology
C700 Molecular Biology, Biophysics and Biochemistry
J700 Industrial Biotechnology
Department: Faculties > Health and Life Sciences > Applied Sciences
Depositing User: Sterghios Moschos
Date Deposited: 03 Aug 2017 11:45
Last Modified: 01 Aug 2021 12:35

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