Nanopore detection of modified RNA and DNA with nucleic acid catalyst-free click chemistry

Characterization of modified RNA is of importance for both the tracking of biological processes and the design and assessment of therapeutic RNA. However, modified RNA identification and quantification across various RNA lengths poses a considerable challenge for sequencing technologies. Herein, we utilized nanopores to detect short and long modified in vitro transcribed RNAs with template-free, catalyst-free nucleic acid click chemistry. We identified biotinylated uridine-modified short RNAs, which were bound to a DNA nanostructure for subsequent nanopore detection. Additionally, we explored the detection of small click modifications, such as azide groups, by optimizing conditions for template-free and catalyst-free click chemistry. Our experiments demonstrate that high concentrations of monovalent salts are essential for achieving quantitative labeling with cyclooctyne oligonucleotides. Furthermore, we successfully decorated long RNAs with azide-modified uridine and mapped their distribution after click reaction with cyclooctyne-oligonucleotides using nanopores. Our study establishes a robust platform for solid-state nanopore characterization of modified RNAs, paving a way towards single-molecule analysis of therapeutic and labeled naturally occurring RNA modifications.