Single-Molecule Characterization of RNA via Nanopore Sensing
Precise characterization of RNA is essential for understanding a wide range of biological processes, studying gene expression, developing new RNA-based therapeutic technologies, and elucidating disease mechanisms. However, current RNA characterization methods often provide an incomplete picture of transcript populations. Some approaches fail to resolve transcript heterogeneity, while those that offer broad overviews typically struggle to capture sequence-specific features or detect chemical modifications which can profoundly influence cellular function and disease. Several RNA characterization techniques still rely on reverse transcription or amplification, introducing enzymatic biases that distort sequence representation and hinder accurate profiling. Given these limitations, there remains a clear need for precise, enzyme-free methods capable of delivering comprehensive RNA analysis. In this thesis, I present a strategy for single-molecule transcript analysis through the assembly of nucleic acid nanostructures directly from the RNA of interest, followed by characterization via solid-state nanopore sensing. I first describe the nanopore observables and nucleic acid parameters relevant to nanostructure characterization. Then, I demonstrate how tandem implementation of nanostructure engineering and nanopore sensing can be used to size transcripts and assess their heterogeneity, applying this method to reveal premature transcription termination within the OriC sequence. Furthermore, I quantify tandem repeats directly on RNA, which are sequences with severe clinical implications that remain difficult to study with current technologies. I also explore critical quality assessment of nucleic acid therapeutics, demonstrating how nanostructure architecture can expand the range of detectable biological markers, such as mRNA cap structures and oligonucleotide PEGylation. Finally, I track co-transcriptional incorporation of modified nucleotides in RNA. Overall, this thesis describes a method capable of reading full-length native transcripts, uncovering structural changes arising from alternative transcript processing, and identifying a variety of structural and chemical transcript modifications at the single-molecule level. This work thus provides an efficient and versatile tool for robust RNA characterization.
Nanopore sensing, RNA therapeutics, RNA:DNA nanotechnology, Solid-state nanopores, Synthetic chemical modifications, Tandem repeats, Transcription
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