Programmable RNA nanostructures enable nanopore detection of co-transcriptionally introduced RNA modifications

Tracking RNA synthesis and metabolic histories requires co-transcriptional incorporation of modified nucleotides. However, identifying the incorporation of modified nucleotides in nascent RNA remains challenging, particularly for short RNAs. Here, we developed a method utilizing solid-state nanopores and DNA:RNA nanostructures to detect modified nucleotide incorporation across different RNA length scales, from short to long RNAs transcribed in vitro. We identified the incorporation of biotin-modified uridine in short RNAs using a DNA nanostructure coupled with nanopore readout. As a proof-of-concept for tracking RNA synthesis, we evaluated the incorporation of azide-modified uridine into long RNAs. To achieve quantitative labeling, we optimized conditions for click chemistry using cyclooctyne-DNA oligonucleotides. Subsequently, we successfully decorated long RNAs with azide-modified uridine and quantified the relative incorporation levels using nanopores. Our study establishes a robust platform for solid-state nanopore characterization of modified nucleotide-containing RNAs, advancing single-molecule analyses of RNA dynamics.