Biology subjects

Largy, E.

Publications and source records attributed to Largy, E..

7 recordsLinked to original sources

bioRxiv2026

Native Hydrogen/Deuterium Exchange Ion Mobility Mass Spectrometry of Structured DNA Oligonucleotides

Hydrogen/deuterium exchange coupled to mass spectrometry (HDX/MS) is a powerful technique to probe nucleic acid secondary structures and dynamics, but its ability to resolve conformers with identical masses remains limited. To overcome this challenge, we integrated ion mobility spectrometry (IMS) into our native HDX/MS workflow, and tested the approach on a variety of model DNA G-quadruplex structures. We show several examples of human telomeric sequence oligonucleotides where complexes of the same mass differ in their collision cross section, and each gas-phase conformational ensembles corresponds to unique solution exchange behaviors, allowing kinetic analysis beyond what is possible with HDX/native MS alone. But we also found examples where several gas-phase populations separated in ion mobility have exactly the same solution exchange behavior, suggesting that conformational rearrangements occur either during electrospray or at later stages in the gas phase. Finally, we show how IMS filtering can be leveraged to distinguish groups of non-specific cation binding on a given conformational ensemble, as indicated by populations with different masses and same exchange rates. These findings establish IMS as an essential tool for complementing HDX/MS in the characterization of structural polymorphism and conformational ensembles in DNA oligonucleotides.

biophysics↗

bioRxiv2025

Unconventional DNA architecture in a dopamine-bound aptamer complex

Aptamers are oligonucleotides that have been selected to bind a particular target. Despite the growing popularity of functional DNA aptamers, there remains limited knowledge of their binding mechanisms as few have been characterized at the atomic level. Here we use NMR spectroscopy to obtain structural details of RKEC1, a shortened version of a DNA aptamer previously reported to bind dopamine. We find that RKEC1 forms a compact structure upon ligand binding that lacks any Watson-Crick duplex regions or G-quadruplex core, in stark contrast to nearly all predicted and observed DNA aptamer folds. The atomic details explain dopamine specificity amongst structurally similar compounds, and the determined DNA fold was used to guide biosensor design. The aptamer structure further suggests that DNA folding can access an extensive conformational landscape reminiscent of RNA, thus expanding the diversity traditionally captured by predictive algorithms.

biochemistry↗

bioRxiv2025

Functional relevance of CASP16 nucleic acid predictions as evaluated by structure providers

Accurate biomolecular structure prediction enables the prediction of mutational effects, the speculation of function based on predicted structural homology, the analysis of ligand binding modes, experimental model building and many other applications. Such algorithms to predict essential functional and structural features remain out of reach for biomolecular. Here, we report quantitative and qualitative evaluation of nucleic acid structures for the CASP16 blind prediction challenge by 12 of the experimental groups who provided nucleic acid targets. Blind predictions accurately model secondary structure and some aspects of tertiary structure, including reasonable global folds for some complex RNAs, however, predictions often lack accuracy in the regions of highest functional importance. All models have inaccuracies in non-canonical regions where, e.g., the nucleic-acid backbone bends or a base forms a non-standard hydrogen bond. These bends and non-canonical interactions are integral to form functionally important regions such as RNA enzymatic active sites. Additionally, the modeling of conserved and functional interfaces between nucleic acids and ligands, proteins, or other nucleic acids remains poor. For some targets, the experimental structures may not represent the only structure the biomolecular complex occupies in solution or in its functional life-cycle, posing a future challenge for the community.

biophysics↗

bioRxiv2025

Helical foldamers as selective G-quadruplex ligands

We investigated the G-quadruplex binding selectivity of short aromatic oligoamide helical foldamers comprising quinoline (Q) and pyridine (P) units. We found that the foldamers prefer parallel G-quadruplex structures, especially when the external G-quartets are sterically accessible. A crystal structure of the tetramer QQPQ with the parallel G-quadruplex formed by dTGGGTTGGGTTGGGTTGGGT shows two quinoline subunits interacting with an external G-quartet through {pi}-stacking, and solution NMR confirms that the foldamer targets the 3 and 5 ends of this G-quadruplex. Foldamers can also selectively target sequence variants of the telomeric sequences containing adenine-to-thymine mutation in the loops. The conformational selectivity of foldamers originates from the bulkiness of oligomers with four or more subunits, which imposes steric restrictions on G-quadruplex binding. The flexibility provided by the pyridine subunits was also key to improve affinity. Mixed quinoline-pyridine foldamers are thus a promising class of selective G-quadruplex ligands, and their unique modular scaffold offers new avenues to further improve their affinity and selectivity.

biophysics↗

bioRxiv2023

A General Framework to Interpret Hydrogen-Deuterium Exchange Native Mass Spectrometry of G-Quadruplex DNA

G-quadruplexes (G4s) are secondary structures formed by guanine-rich oligonucleotides involved in various biological processes. However, characterizing G4s is challenging because of their structural polymorphism. Here, we establish how hydrogen-deuterium exchange native mass spectrometry (HDX/MS) can help to characterize G4 structures and dynamics in solution. We correlated the time range of G4 exchange to the number of guanines involved in inner and outer tetrads. We also established relationships between exchange rates, number of tetrads, bound cations, and stability. The use of HDX/native MS allows for the determination of tetrads formed and assessment of G4 stability at a constant temperature. A key finding is that stable G4s exchange through local fluctuations (EX2 exchange), whereas less stable G4s also undergo exchange through partial or complete unfolding (EX1 exchange). Deconvolution of the bimodal isotope distributions resulting from EX1 exchange provides valuable insight into the kinetics of folding and unfolding processes, and allows one to detect and characterize transiently unfolded intermediates, even if scarcely populated. HDX/native MS thus represents a powerful tool for a more comprehensive exploration of the folding landscapes of G4s. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/554600v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@17a4462org.highwire.dtl.DTLVardef@1fb0f3org.highwire.dtl.DTLVardef@9a5b07org.highwire.dtl.DTLVardef@1ce417e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

DNA G-quadruplexes for native mass spectrometry in potassium: a database of validated structures in electrospray-compatible conditions

G-quadruplex DNA structures have become attractive drug targets, and native mass spectrometry can provide detailed characterization of drug binding stoichiometry and affinity, potentially at high throughput. However, the G-quadruplex DNA polymorphism poses problems for interpreting ligand screening assays. In order to establish standardized MS-based screening assays, we studied 28 sequences with documented NMR structures in (usually 100 mM) K+, and report here their circular dichroism (CD), melting temperature (Tm), NMR spectra and electrospray mass spectra in 1 mM KCl/100 mM TMAA. Based on these results, we make a short-list of sequences that adopt the same structure in the MS assay as reported by NMR, and provide recommendations on using them for MS-based assays. We also built an R-based open-source application to build and consult a database, wherein further sequences can be incorporated in the future. The application handles automatically most of the data processing, and allows generating custom figures and reports. The database is included in the g4dbr package (https://github.com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4.github.io/G4_database.html).

biophysics↗

bioRxiv2019

Native Hydrogen/Deuterium Exchange Mass Spectrometry of Structured DNA Oligonucleotides

Although solution hydrogen-deuterium exchange mass spectrometry (HDX/MS) is well-established for the analysis of the structure and dynamics of proteins, it is currently not exploited for nucleic acids. Here we used DNA G-quadruplex structures as model systems to demonstrate that DNA oligonucleotides are amenable to in-solution HDX/MS in native conditions. In trimethylammonium acetate solutions and in soft source conditions, the protonated phosphate groups are fully back-exchanged in the source, while the exchanged nucleobases remain labeled without detectable back-exchange. As a result, the exchange rates depend strongly on the secondary structure (hydrogen bonding status) of the oligonucleotides, but neither on their charge state nor on the presence of non-specific adducts. We show that native mass spectrometry methods can measure these exchange rates on the second to the day time scale with high precision. Such combination of HDX with native MS opens promising avenues for the analysis of the structural and biophysical properties of oligonucleotides and their complexes.

biophysics↗