# Exhaustive de novo sequencing of every RNA in a sample by a layer-by-layer mass spectrometry ladder intensity approach.

> **NIH NIH R41** · DIRECTSEQ BIOSCIENCES, INC. · 2024 · $215,088

## Abstract

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.

## Key facts

- **NIH application ID:** 11003488
- **Project number:** 1R41HG014125-01
- **Recipient organization:** DIRECTSEQ BIOSCIENCES, INC.
- **Principal Investigator:** Shenglong Zhang
- **Activity code:** R41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $215,088
- **Award type:** 1
- **Project period:** 2024-09-25 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11003488

## Citation

> US National Institutes of Health, RePORTER application 11003488, Exhaustive de novo sequencing of every RNA in a sample by a layer-by-layer mass spectrometry ladder intensity approach. (1R41HG014125-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/11003488. Licensed CC0.

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