Nanopore detection of single-nucleotide RNA mutations and modifications with programmable nanolatches

RNA mutations and modifications have been implicated in a wide range of pathophysiologies. However, current RNA detection methods are hindered by data complexity and error-prone protocols, restricting their widespread use. Here we present a solid-state nanopore-based approach, RNA single-nucleotide characterization and analysis nanolatch (RNA-SCAN) system, which simplifies the detection of nucleotide mutations and modifications in RNA with high resolution. Using phage RNA as a template, we tested multiple sequences and chemical modifications on nanolatches, allowing the detection of mismatches caused by nucleotide mutations through significant changes in positive event ratios using single-molecule nanopore measurements. This approach is also sensitive to modifications that either strengthen or weaken the interaction between the target RNA sequence and the nanolatch. As a proof-of-concept, we demonstrate successful discrimination of Escherichia coli and Salmonella spp. from total RNA based on nucleotide variations in their 16S rRNA, as well as quantification of different Salmonella spp. and detection of m⁵C1407 modification on E. coli 16S rRNA. The RNA-SCAN approach demonstrates the feasibility of combining RNA/DNA hybrid nanotechnology with nanopore sensing and diagnosing RNA-related health conditions.