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

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.