Programmable RNA Nanostructures Enable Nanopore Detection of Cotranscriptionally Introduced RNA Modifications

Tracking RNA synthesis and metabolic histories requires cotranscriptional incorporation of modified nucleotides. However, identifying the incorporation of modified nucleotides into nascent RNA remains challenging, particularly for short RNAs. In this work, 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 <i>in vitro</i>. We identified the incorporation of biotin-modified uridine in short RNAs using a DNA nanostructure coupled with a 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.