De novo direct sequencing of small therapeutic RNAs by layer-by-layer intensity-resolved mass spectrometry.

The rapid growth of RNA-based therapeutics demands accurate sequencing of all RNA species, including minor and modified variants. Conventional LC-MS/MS typically confirms only a predefined target sequence rather than determining RNA sequences de novo from the analyzed sample, thereby overlooking coexisting impurities and modifications. Here, we present 3D NGMS-Seq, a three-dimensional next-generation mass spectrometry-based sequencing platform for de novo direct sequencing of mixed RNA samples with essentially 100% sequence accuracy. This method incorporates MS intensity into traditional 2D mass-retention time (tR) analysis and introduces a nested algorithm that aligns ladder fragment intensities with parent RNA abundances for computational separation. Controlled acid hydrolysis produces RNA ladder fragments, which are segregated into mass-intensity-tR layers. Within each layer, short reads are generated de novo by sequentially base-calling each nucleotide, canonical or modified, from mass differences between adjacent ladder fragments and subsequently assembled into full-length RNA sequences. Guided by hydrolysis kinetics and statistical modeling, 3D NGMS-Seq accurately sequences synthetic siRNA, miRNA, and CRISPR/Cas9 sgRNAs, reveals unexpected low-abundance RNA impurities, and resolves subtle methylation ambiguities (Um versus mU; Am versus mA), while providing a quantitative profile of each RNA's relative abundance and site-specific modifications. By enabling direct, unbiased sequencing of heterogeneous RNAs without prior sequence knowledge, 3D NGMS-Seq addresses key limitations of current RNA analysis and provides a powerful tool to aid small RNA drug development, quality control, and regulatory validation.