Molecular Encoding during Amplification Enables Multiplex Nanopore Profiling of Small Noncoding RNAs

Many cancers are characterized by the coordinated dysregulation of multiple small noncoding RNAs (sncRNAs), yet fluorescence-based assays such as quantitative reverse transcription PCR (qRT-PCR) fundamentally limit multiplexing. Here, we report an amplification-encoded nanopore sensing strategy in which sncRNA identity is written during RT-PCR amplification and decoded by single-molecule nanopore sensing without chemical labeling or optical detection. Target sncRNAs are converted into stem-loop amplicons whose electrical signatures are modulated by the nucleotides at the 3' terminus of sncRNA, allowing for the encoding of many distinct sncRNA identities and enabling single-nucleotide discrimination within the highly homologous Let-7 family. As a demonstration, we concurrently analyzed gastric cancer-associated microRNAs and tRNA-derived small RNAs, achieving classification accuracies exceeding 97% with machine-learning-assisted decoding. This method also enables absolute quantification of multiple sncRNAs simultaneously through capture-efficiency correction and an internal reference sequence. This amplification-encoded nanopore strategy provides a scalable framework for multiplex single-molecule analysis of diverse sncRNAs and highlights its potential for liquid biopsy applications.