Direct RNA sequencing holds great promise for the de novo identification of RNA modifications at single-coordinate resolution; however, interpretation of raw sequencing output to discover modified bases remains a challenge. Using Oxford Nanopore's direct RNA sequencing technology, we developed a random forest classifier trained using experimentally detected N 6-methyladenosine (m 6 A) sites within DRACH motifs. Our software MINES (m 6 A Identification using Nanopore Sequencing) assigned m 6 A methylation status to more than 13,000 previously unannotated DRACH sites in endogenous HEK293T transcripts and identified more than 40,000 sites with isoform-level resolution in a human mammary epithelial cell line. These sites displayed sensitivity to the m 6 A writer, METTL3, and eraser, ALKBH5, respectively. MINES (https:// github.com/YeoLab/MINES.git) enables m 6 A annotation at single coordinate-level resolution from direct RNA nanopore sequencing.

Highlights• STAMP identifies binding sites of full-length RBPs by C-to-U RNA editing.• STAMP coupled to long read sequencing reveals isoform specific RBP targets.• STAMP allows cell-type specific and multiplexed-RBP target identification in single cells.• STAMP with ribosome subunits allows detection of ribosome association at single-cell level.

MicroRNAs (miRNA) play critical roles in human development and disease. As such, the targeting of miRNAs is considered attractive as a novel therapeutic strategy. A major bottleneck toward this goal, however, has been the identification of small molecule probes that are specific for select RNAs and methods that will facilitate such discovery efforts. Using pre-microRNAs as proof-of-concept, herein we report a conceptually new and innovative approach for assaying RNA-small molecule interactions. Through this platform assay technology, which we term catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a method that can be implemented in high throughput, is virtually free of false readouts, and is general for all nucleic acids. Through cat-ELCCA, we envision the discovery of selective small molecule ligands for disease-relevant miRNAs to promote the field of RNA-targeted drug discovery and further our understanding of the role of miRNAs in cellular biology.

Catalytic enzyme-linked click-chemistry assays (cat-ELCCA) are an emerging class of biochemical assay. Herein we report on expanding the toolkit of cat-ELCCA to include the kinetically superior inverse-electron demand Diels-Alder (IEDDA) reaction. The result is a technology with improved sensitivity and reproducibility, enabling automated high-throughput screening.