RACE-Seq identifies the Argonaute-2 cleavage products of RNA interference-based oligonucleotides

The recent announcement of the first successful Phase III clinical trial of a RNA interference (RNAi)-based therapeutic is a major achievement in the field. Synthetic RNAi therapeutic oligonucleotides are either first cleaved by Dicer or incorporate directly into the Argonaute-2 RNA-induced silencing complex (AGO2-RISC) and directs the protein complex to homologous RNA. Cleavage of target RNA occurs opposite bases 10-11 when counting from the 5’ end of the hybridized siRNA guide strand. The capture and identification of these cleaved products by 5’ Rapid Amplification of cDNA Ends and Sanger sequencing remains the gold standard for confirming Argonaute-2 mediated RNAi cleavage.
Next Generation Sequencing of 5’ RACE has brought new insights into the biological activity of RNAi-based oligonucleotides. This work currently represents the largest undertaking using RACE-Seq to investigate AGO2-RISC-mediated activity. RACESeq reported the expected RISC-cleaved product for each of the oligonucleotides investigated. Additionally, RACE-Seq analysis revealed that some of the oligonucleotides could be processed into multiple active siRNA molecules. Analysis of the activity of a Dicer substrate siRNA targeting transthyretin revealed that this molecule by-passed Dicer processing but still induced RNAi activity. In examining RACE-Seq peak profiles, an on-target mechanism of action (MOA) for up to four active siRNA derived from siRNA19 is proposed. The shRNA19 RACE-Seq assay predicted that this hairpin molecule probably exists as two distinct forms, one with a 7-nucleotide loop and the other with a 5-nucleotide loop.
The project also focused on optimising the library preparation, data filtering and data presentation for RACE-Seq. A simplified, low computation data analysis pipeline was designed and used to align the filtered dataset to a reference sequence and to count the 5’ ends. RACE-Seq is presented as a suitable solution for investigating, discriminating and quantifying specific RNA cleavage events and visualizing evidence for an on-target MOA of RNAi based oligonucleotide therapeutics.