A reference-guided iterative nanopore workflow improved for quality control of modified therapeutic RNAs.
Evidence
The evidence is a sequencing-methods study testing the approach on artificially synthesized molecules, real-world molecules, vaccine mRNAs, and human tRNA-based BioRNA carriers for RNAi agents.
Caveat
The results support analytical quality control of RNA sequence purity, integrity, and modification hotspots, but the abstract does not test therapeutic efficacy or clinical outcomes.
Simplified
Nucleotide modifications deviate nanopore sequencing readouts, therefore generating artifacts during the of sequence backbones. Here, we present a reference-guided, iterative approach to polish modification-disturbed basecalling results. We show that such an approach is uniquely suitable for training biomolecule-specific high-accuracy basecallers, by improving the basecalling of both artificially-synthesized and real-world molecules. With demonstrated efficacy and reliability, we exploit the approach to precisely basecall therapeutic RNAs consisting of artificial or natural modifications. We first analyzed vaccine mRNAs, which are artificially modified to promote stability and reduce immunogenicity. Specifically, we quantified the sequence purity and integrity, the two most important quality metrics to be controlled during mRNA vaccine production. We also analyzed BioRNAs, which are human tRNA-based carriers for therapeutic RNA interference (RNAi) agents. Specifically, we examined modification hotspots, which are naturally incorporated in vivo during BioRNA production and essential for therapeutic efficacy. Our analysis expands the scope of therapeutic RNA quality control, from the conventional sequence-level to the current modification status-level.
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Competing interests: H.D. has a patent application related to this work (Hongxu Ding, An Iterative Approach to Polish the Nanopore Sequencing Basecalling for Therapeutic RNA Quality Control, US Provisional Patent Application No. 63/693,352). Z.W. has received travel expenses from Oxford Nanopore Technologies to speak at the 2025 RNA annual meeting. Other authors declare no competing interests.
