Nanopore detection of single-nucleotide RNA mutations and modifications with programmable nanolatches.
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Springer Nature
https://doi.org/10.1038/s41565-025-01965-6
https://doi.org/10.1038/s41565-025-01965-6
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Funder: UK Research and Innovation (UKRI) under the UK government’s Horizon Europe funding guarantee (EP/X023311/1)
Funder: China Scholarship Council (CSC); doi: https://doi.org/10.13039/501100004543
Funder: Cambridge Commonwealth, European and International Trust (Cambridge Commonwealth, European & International Trust); doi: https://doi.org/10.13039/501100003343
Funder: Fundamental Research Funds for the Central Universities of China (Grant No. DUT24YG211) Liaoning Provincial Science and Technology Joint Fund (2023JH2/101800039 and 2023JH2/101800037)
Funder: Herchel Smith Fund for a Postdoctoral Fellowship
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.
Funder: China Scholarship Council (CSC); doi: https://doi.org/10.13039/501100004543
Funder: Cambridge Commonwealth, European and International Trust (Cambridge Commonwealth, European & International Trust); doi: https://doi.org/10.13039/501100003343
Funder: Fundamental Research Funds for the Central Universities of China (Grant No. DUT24YG211) Liaoning Provincial Science and Technology Joint Fund (2023JH2/101800039 and 2023JH2/101800037)
Funder: Herchel Smith Fund for a Postdoctoral Fellowship
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.