Short DNA and RNA sequences called oligonucleotides can fold into complex shapes that determine their function, but scientists lack a fast, reliable way to map those shapes in solution. Current methods for studying oligonucleotide structure each have serious limits: X-ray crystallography struggles with flexible molecules, cryo-electron microscopy cannot see small ones, and nuclear magnetic resonance spectroscopy is slow and requires large samples. This leaves a blind spot for many biologically important sequences—including aptamers that bind targets like antibodies, G-quadruplexes involved in gene regulation, and potential drug or vaccine candidates. The researchers aim to fill that gap by adapting hydrogen-deuterium exchange mass spectrometry (HDX-MS), a technique already routine for proteins, to work on oligonucleotides for the first time. If successful, this fundamental science project will give researchers a new tool to probe how oligonucleotides fold, how they interact with proteins or small molecules, and how those interactions change with sequence modifications. That understanding could guide the design of oligonucleotide-based drugs and vaccines, or help explain how natural regulatory RNAs work. The work is exploratory—establishing basic methodology on simple test sequences—but mirrors how HDX-MS for proteins became a key enabler in biopharmaceutical development.
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Oligonucleotides (OGNs) are an important class of biomolecules, but until recently somewhat neglected in structural biology research. Well-known functions range from storage of inherited information to gene regulation; but short DNA and RNA sequences can also provide interaction motifs for proteins and antibody-like high-affinity binding e.g. for small molecules (aptamers). Recently there has been a surge of interest in OGNs and their derivatives for potential use as drugs and vaccines, but also as targets of pharmaceutical intervention. Following the hypothesis that structure informs function, we need to understand fundamental principles of structure-function relationships for OGNs, which can also be dynamic and partly unstructured. Currently our insights and toolkit are limited: many of the interesting sequences are either too small for cryo-EM or too dynamic to easily yield x-ray structures. NMR spectroscopy has delivered valuable insights, e.g., for single-strand DNA aptamer and G-quadruplex sequences, but is neither fast nor routine and requires considerable expertise and amounts of sample. Mass spectrometry (MS) has become a breakthrough technology for biomolecular characterization in the last 25+ years. It can link information on small changes in the protein sequence (such as post-translational modifications or mutations) with higher-order structure (folding) and interactions, and deliver exceptional insights into intrinsic disorder and lipid interactions. While not actually providing high resolution images, structural MS approaches such as native MS, ion mobility, chemical crosslinking and labeling, and hydrogen-deuterium exchange (HDX) can nevertheless characterize dynamic and heterogeneous distributions of conformational states and interaction stoichiometries, at high sensitivity and with little bias. They are often used in conjunction with other approaches including computational modeling. For OGNs, some work has been done with native MS and ion mobility (which determines global size and shape) to study structure and interactions of aptamers, G-quadruplexes and DNA nanostructures, but there is much left to understand and explore. Here we propose to develop and apply hydrogen-deuterium exchange (HDX)-MS approaches for the study of dynamic oligonucleotide structures of biological interest. This work aims at gaining fundamental insights into OGN folding and interactions to guide pharmaceutical strategies. We will pioneer HDX-MS for OGN structure, an almost entirely unexplored approach. For proteins, the technique has become a key enabler for studying binding interfaces, allosteric effects and biopharmaceutical design, with substantial recent investment across research institutions in academia and industry. No equivalent methodology exists yet for OGNs due to lack of knowledge of exchangeable residues and their timescales, quenching conditions and back exchange, and suitable digestion strategies and chromatographies. In order to address these challenges, we will initially explore the ability of HDX-MS to detect defined structures (single/double-stranded DNA, mismatches/kinks and crosslinks, stem-loops etc.), using simple short test sequences. The approach which we will take to reach the aims detailed above includes three objectives, with work packages built around them: 1. Establish comprehensive OGN HDX-MS methodology 1.1 "Global" HDX of native OGNs with UVPD fragmentation 1.2 In-line digested LC-MS analysis to mirror protein HDX 2. Demonstrate structural characterization using simple motifs 2.1 Detection of single vs. double-stranded sequences 2.2 Aptamer folding and ligand interactions 2.3 Characterization of G quadruplexes 3. Apply OGN HDX-MS to biological questions 3.1 Collaboration with Wu (Leeds) 3.2 Collaboration with Dillingham (Bristol)
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