An RNA sequence and all its diverse modifications constitutes the true informational content of RNA. Defects in RNA modifications account for >100 human diseases, affecting millions of Americans, including those with breast cancer, skin cancer, and lung cancer. Despite its significance, our understanding of RNA sequence and modification diversity remains limited. Current sequencing technologies only partially capture RNA sequences and modifications, leaving gaps in our understanding. For instance, the exact count of unique RNA sequences in a sample remains elusive. NGS-based RNA sequencing methods require converting RNA to cDNA, and such indirect methods can not accurately reflect the original RNA content. While nanopore-based RNA sequencing permits direct RNA sequencing, it relies on ligating RNAs to sequence adapters, a process with variable yields ranging from 30% to 90%. Consequently, any RNA failing this ligation remains unsequenced, leading to potential underrepresentation. Furthermore, sequencing all RNA modifications is challenging due to the presence of >170 unique nucleotide modifications. NGS-based RNA sequencing technologies, relying on cDNA synthesis, often neglect or bias towards certain RNA modifications, thus failing to deliver a comprehensive profile of RNA modifications. This issue is exacerbated by the heterogeneous nature and quantification difficulties associated with partial RNA modifications. To address these challenges, DSI has innovated next-generation mass spectrometry-based sequencing (NGMS-Seq) methods. Utilizing two-dimensional (2D) mass-retention time ladders instead of MS/MS fragmentation, NGMS-Seq has demonstrated the potential to sequence specific tRNAs de novo and simultaneously sequence and quantify all nucleotide modifications without bias. In this application, we will 1) further develop NGMS-Seq into an exhaustive de novo RNA sequencing method, which not only directly identifies but also sequences every RNA species completely in a sample without any omission, and provides a comprehensive modification landscape for each RNA without bias, and 2) test exhaustive sequencing of synthetic RNA therapeutics (Aim 1) and human cancer tRNAs (Aim 2) for comprehensive RNA sequence and modification profiling. This advancement could revolutionize our ability to fully understand RNA's sequence and modification diversity, crucial for RNA drug development and functional studies.