Work

Rapid Exonuclease‐Based De Novo Sequencing Strategy for Long Oligonucleotides via Liquid‐Phase Ion Trap Coupled With Mass Spectrometry

Year: 2026

Type: article

Abstract: The sequencing of synthetic oligonucleotides is fundamental to life sciences and therapeutics, yet current analytical approaches present inherent technical drawbacks of varying degrees. Hybridization-based techniques cannot sequence the oligonucleotide primers they rely on, while the accuracy of mass spectrometry (MS)-based techniques significantly diminishes as fragment length increases. Here, we introduce a liquid-phase ion trap (LPIT) coupled with electrospray ionization (ESI)-MS as a solution for rapid, high-fidelity oligonucleotide sequencing. The LPIT utilizes a strong electric field to efficiently dissociate confounding metal cation adducts prior to MS analysis, resulting in cleaner spectra and enhanced signal intensity. This "desalting-first" approach enables direct, rapid oligonucleotide analysis, achieving mass measurement accuracy comparable to benchmark liquid chromatography-ESI-MS (LC-ESI-MS) platforms. By integrating this platform with an exonuclease digestion workflow, we achieved full sequence coverage of oligonucleotides in under 30 min, a dramatic improvement in speed over existing methods. This work establishes LPIT-ESI-MS as a powerful platform for the direct and rapid sequencing of long oligonucleotides, addressing a critical unmet need in biotechnology and drug development.

Source: Advanced Science

DOI: 10.1002/advs.77313

Authors: Song Lu, Jie Hong, Xinyan Fu, Di Wang, Yunhua Gao, Wei Xu

Institutions: National Institute of Metrology, Beijing Institute of Technology

Language: English

Topic: Mass Spectrometry Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: gold

Estimated APC paid: $6,730

Funders: National Natural Science Foundation of China, National Key Research and Development Program of China