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

Abstract

Low allelic and clonal variability among endogenous 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) analogs 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 semiquantitatively report the impact of RNAi hybridization site mismatches from the target perspective. The analysis shows that Argonaute 2 has a substitution-specific, 3- to 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.

Computational search of hybrid human/SARS-CoV-2 dsRNA reveals unique viral sequences that diverge from those of other coronavirus strains

“…In the past, RNA duplex with mismatches have been tested for their efficiency to promote RNA target degradation by Ago2. In some examples a single mismatch in a 19-bases length sequence can prevent and/or fragilize duplex formation by creating thermodynamic asymmetry ( Parrish et al, 2000 ; Theotokis et al, 2017 ). In other examples these heteroduplexes can still promote RNAi activity on mRNA targets, but with moderately to drastically reduced effectiveness when they are compared to perfect RNA pairing.…”

RACE-SEQ and Population-Wide Polymorphism Susceptibility Testing for Endonucleolytically Active, RNA-Targeting Therapeutics

“…Furthermore, as additional data emerged on the method from multiple laboratories, it became clear that the extent of information returned from this analytical approach was a function of the biology of the experimental setup, and the data analysis pipeline implemented. Thus, parameters such as the mutation rate of the RNA target (tissue RNA (5-7), endogenous targets in cell culture (8), viral RNA or replicon RNA in cell culture (4,9,10)), depth of sequencing and breadth of sequencing (4) orchestrate the observed outcomes, including predictive pharmacogenomic assessment of drug efficacy against targets mismatched to the drug (RACE-SEQ-MM: (9)).…”

“…45. Bowtie v.2.0.0 or later is not suited to RACE-SEQ due to its mismatch-tolerating read alignment methodology around alignment seed length (seeTheotokis et al 2017).…”