High-Resolution Pseudouridine and m6A Sequencing Through Advanced Nanopore Adduct Analysis

Project Summary The landscape of transcriptomic and epitranscripotmic research is on the cusp of a revolution, with RNA modifications playing pivotal roles in understanding gene expression, regulation, and the manifestation of diseases. Among these modifications, pseudouridine (Ψ) and N6-methyladenosine (m6A) are of significant interest due to their prevalence and impact on RNA stability, protein synthesis, and cellular stress responses. However, current RNA sequencing technologies have low accuracy and precision in detecting RNA modifications and are only able to inspect one modification at a time, thus limiting our understanding of their biological role, and our ability to develop RNA-based diagnostics and therapeutics. RNA direct nanopore sequencing is an attractive approach to sequencing many modifications at once. However, the current commercial nanopore sequencer is limited by a high error rate in detecting Ψ and m6A because Ψ and m6A fail to yield quantitatively unique sequencing signatures relative to their parent nucleotides and other RNA modifications; consequently, the false discovery rate is too high for any clinical application and single molecule analysis is not possible. To fully understand the roles of these crucial epitranscriptomic modifiers, it is essential to accurately and concurrently sequence both modifications within a single experiment. This Phase I STTR project, a collaborative effort between academia and industry, is set to develop the first technology capable of high-accuracy sequencing of Ψ and m6A, by merging Distinguished Professor Cynthia J. Burrows' innovative bisulfite adduct methodology with Electronic BioSciences’ (EBS) enzyme-free, multipass nanopore nucleic acid characterization technology. This innovative approach aims to accurately identify Ψ and m6A modifications with unprecedented precision (>99.9%), overcoming the limitations of current sequencing methods. The project is structured around two primary aims: 1) The analytical validation of sequencing RNA with Ψ and m6A modifications using our novel technology, and 2) The application of this technology to sequence mRNA from renal cell carcinoma cells, providing insights into the epitranscriptomic landscape of this cancer. Expected outcomes include the establishment of the first sequencing platform capable of high-precision and simultaneous mapping of Ψ and m6A modifications, offering a significant advancement in the field of RNA research. This technology will not only enhance our understanding of gene regulation and disease mechanisms but also pave the way for novel diagnostics and personalized RNA-based therapies. Through its approach and potential applications, this project represents a significant step forward in the quest to unravel the complexities of the RNA epitranscriptome, with broad implications for medical science and therapeutics. Ultimately, this project embodies a strategic effort to advance our capability to study RNA modifications through innovative technology that will be poised to deliver solutions to sequence many other chemical changes to RNA as well.