# High-throughput Direct Sequencing and Quantitative Mapping of RNA Modifications using Mass Spectrometry.

> **NIH NIH R41** · DIRECTSEQ BIOSCIENCES, INC. · 2024 · $406,500

## Abstract

Sequencing RNA modifications is challenging due to the existence of over 170 unique chemical
nucleotide alterations. tRNAs, constituting about 25% of cellular RNA and decoding 61 mRNA
codons, often have 8-14 modifications each. These modifications are central to functional
diversity, cellular regulation, and diseases such as cancer and neurological disorders.
Unbiased, comprehensive sequencing of RNA modifications is vital for understanding these
complex processes. Current techniques, including Next-Generation Sequencing (NGS), depend
on complementary DNA (cDNA) intermediates, failing to capture the complete tRNA
modification profile. They often overlook or bias certain types, and are not suitable for tRNAs,
which have reverse-transcriptase-blocking nucleotide modifications. The heterogeneity of tRNA
modifications, with over 100 identified types showing varying modification levels, adds
complexity. Although nanopores show potential for sequencing all RNA modifications, training
over 170 different standard nucleotide modifications while preserving unique signature
electronic signals presents formidable challenges. Mass spectrometry (MS), despite its ability to
characterize RNA modifications without bias, has limitations. Conventional MS methods like
tandem MS (MS2) lose essential details about modified nucleotide location and co-occurrence,
and the complexity of its spectra obstructs high-throughput sequencing of intricate tRNAs. To
counter these challenges, we've developed next-generation mass spectrometry-based
sequencing (NGMS-Seq) methods. Utilizing two-dimensional (2D) mass-retention time ladders
instead of MS2 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, our aims are to: 1) advance NGMS-Seq toward high throughput for
direct sequencing of various tRNA samples, including physiologically relevant ones, and 2)
enhance technologies to quantitatively map tRNA modifications site-specifically and track their
dynamic changes within a melanoma model. Our refined techniques, initially tailored for tRNA,
have the potential to extend to longer RNAs and small noncoding RNAs. Through our
commercialization initiatives, we aim to make NGMS-Seq widely accessible, providing
comprehensive RNA sequence and modification data, thereby offering vital insights into RNA-
associated diseases.

## Key facts

- **NIH application ID:** 10920130
- **Project number:** 1R41HG013624-01
- **Recipient organization:** DIRECTSEQ BIOSCIENCES, INC.
- **Principal Investigator:** Shenglong Zhang
- **Activity code:** R41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $406,500
- **Award type:** 1
- **Project period:** 2024-09-18 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10920130

## Citation

> US National Institutes of Health, RePORTER application 10920130, High-throughput Direct Sequencing and Quantitative Mapping of RNA Modifications using Mass Spectrometry. (1R41HG013624-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10920130. Licensed CC0.

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