High Precision Quantification of small RNA Slicing Activity - Native Index Ligation-based Targeted Degradome Sequencing (NIL-TDS)

RNA interference (RNAi) is an effective and precise regulatory mechanism in eukaryotes in which small RNAs mediate endonucleolytic slicing of complementary target mRNAs. Despite the potential of RNAi for human therapeutics and crop bio-protection, analytical platforms to quantitatively validate the slicing activities of small RNAs remain limited. Here, we present NIL-TDS, a cost-effective method that combines RNA ligase-mediated PCR with Nanopore Sequencing for direct, rapid, and high-resolution detection and quantification of sRNA-mediated slicing events. Using NIL-TDS, we quantitatively detected minute changes in Ath -mir400 mediated slicing of PPR1 in heat and salt stressed Arabidopsis plants. We further demonstrate the broader applicability of NIL-TDS by detecting rare slicing events in a mammalian system. Of relevance for malignancy of certain cancers, and tumor progression and metastasis, NIL-TDS further confirmed miR-196-HOXB8 interactions in lung cancer cells and discovered a novel miR-7162-HOXA10-AS slicing site. These findings demonstrate the sensitivity of NIL-TDS for uncovering small RNA mediated regulatory mechanisms of gene expression and disease progression in eukaryotes.