Biology subjects

Rosu, F.

Publications and source records attributed to Rosu, F..

6 recordsLinked to original sources

bioRxiv2025

Activation of oligonucleotide polyanions using collisions, electrons and photons in a timsOmni platform

We describe here various ion activation experiments realized in the Omnitrap platform integrated on the timsOmniTM mass spectrometer for the analysis of oligonucleotides in the negative ion mode. The activation methods include resonance collision-induced dissociation (RCID), electron detachment dissociation (EDD), infrared laser multiple-photon activation (IRMPD) and UV laser photodissociation (UVPD). Special emphasis is given to EDD, either as a standalone technique or in conjunction with vibrational re-activation of the ion radicals. We describe EDD on standard 6-mer DNA sequences that have been extensively characterized on other instruments, followed by a comparison of several activation approaches for the phosphorothioate-based oligonucleotide therapeutics Fomivirsen, and concluding with the fragmentation analysis of 46-mer DNA and RNA. EDD alone already provides excellent sequence information on Fomivirsen, but MS3 combinations such as EDD-RCID or EDD-IRMPD proved even more effective, including for the 46-mer DNA (less prone to fragmentation than RNA) at a relatively low charge state. The diversity of ion activation combinations available on the Omnitrap platform is demonstrated by an MS4 experiment investigating the fate of a* and z* radical fragments produced by EDD. TOC graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/687689v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@534c32org.highwire.dtl.DTLVardef@af8ab0org.highwire.dtl.DTLVardef@ba2c3dorg.highwire.dtl.DTLVardef@ae9196_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Native Ion Mobility-MS Reveals the Bead Ejection Mechanism in Multi-Domain Nucleic Acids

Understanding how biomolecules acquire their charge and retain their solution conformation during electrospray ionization (ESI) is crucial for native mass spectrometry (native MS) interpretation. Here, we examine the charging and gas phase conformation of nucleic acid constructs comprising folded G-quadruplex "beads" linked by unstructured polythymine regions. Under physiological ionic strength, these oligonucleotides exhibit a multimodal charge-state and collision cross section distribution, revealing multiple conformational ensembles, in contrast to the unimodal profiles typically observed for shorter oligonucleotides. Native MS observations for intermediate charge states are compatible with ion production via the recently proposed bead ejection scenario, in addition to the charge residue scenario for low charge states and chain ejection for the highest charge states or for sequences with thymine overhangs on both ends. The preservation of the local structures in ions charged above the Rayleigh limit helps to infer the presence of folded subunits. The position of the G-quadruplex subunit and ionic strength governs the charging and retention of G-quadruplex folded regions. Our findings broaden the existing conceptual framework underpinning nucleic acid ionization. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/667413v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@42d7c3org.highwire.dtl.DTLVardef@63445dorg.highwire.dtl.DTLVardef@11d7056org.highwire.dtl.DTLVardef@1ba48bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Negative Electrospray Supercharging Mechanisms of Oligonucleotide Single Strands and G-quadruplexes

When sprayed from physiological ionic strength, nucleic acids typically end up with low levels of charging and in compact conformations. Increasing the electrospray negative charging of nucleic acids while preserving the native non-covalent interactions can help distinguish solution folds by ion mobility mass spectrometry. To get fundamental insight into the supercharging mechanisms of nucleic acids in the negative mode, we studied model G-quadruplex structures and single strand controls in 100 mM ammonium acetate. We found that adding 0.4% propylene carbonate, 0.4% sulfolane or 0.1% m-NBA induces native supercharging through the charged residue mechanism. However, although 0.4% m-NBA shows the highest supercharging ability, it induces unwanted unfolding of solution-folded G-quadruplexes. The supercharging effect resembles the effect of lowering the ionic strength, which could be explained by partial neutralization of the ampholytes when droplets become more concentrated in their non-aqueous components. The supercharging ability ranks: PC < sulfolane < m-NBA. m-NBA adducts to G-quadruplexes with high charge states confirms that the supercharging agent interacts directly with DNA. Surprisingly, in presence of supercharging agents, more negative charge states also bear more alkali metal ion adducts. This suggests that native supercharging results from larger droplets evaporating to the charged residue, leading to higher concentration of both the supercharging agent and of alkali counterions. However, when negative charge carriers from the electrolyte become too rare, chain ejection accompanied by denaturation, and hence non-native supercharging, can become predominant.

biophysics↗

bioRxiv2019

Probing Ligand and Cation Binding Sites in G-Quadruplex Nucleic Acids by Mass Spectrometry and Electron Photodetachment Dissociation Sequencing

Mass spectrometry provides exquisite detail on ligand and cation binding stoichiometries with a DNA target. The next important step is to develop reliable methods to determine the cation and ligand binding sites in each complex separated by the mass spectrometer. To circumvent the caveat of ligand derivatization for cross-linking, which may alter the ligand binding mode, we explored a tandem mass spectrometry (MS/MS) method that does not require ligand derivatization, and is therefore also applicable to localize metal cations. By obtaining more negative charge states for the complexes using supercharging agents, and by creating radical ions by electron photodetachment, oligonucleotide bonds become weaker than the DNA-cation or DNA-ligand noncovalent bonds upon collision-induced dissociation of the radicals. This electron photodetachment (EPD) method allows to locate the binding regions of cations and ligands by top-down sequencing of the oligonucleotide target. The very potent G-quadruplex ligands 360A and PhenDC3 were found to replace a potassium cation and bind close to the central loop of 4-repeat human telomeric sequences.

biophysics↗

bioRxiv2018

Thermal Denaturation of DNA G-Quadruplexes and their Complexes with Ligands: Thermodynamic Analysis of the Multiple States Revealed by Native Mass Spectrometry

As the idea that G-quadruplex nucleic acid structures are involved in cellular processes is gaining support, it becomes important to develop ligands that specifically target G-quadruplexes. However, ligand design is complicated because there are multiple G-quadruplex target sequences, some sequences are polymorphic, and very few ligand-quadruplex structures in solution were solved to date. Further, structure alone does not reveal the driving forces for ligand binding. To know why a ligand binds, the thermodynamics of binding must be characterized. Electrospray mass spectrometry makes it possible to detect and quantify each specific stoichiometry in terms of number of strands, number of specific cations, and number of ligands, and thus allows one to simultaneously determine the equilibrium constants for the formation of each complex. We designed and built a temperature-controlled nano-electrospray source to monitor thermal denaturation by mass spectrometry (\"MS-melting\"). We studied the thermal denaturation of G-quadruplexes, including the c-myc promoter and several telomeric sequence variants, and their complexes with popular ligands (Phen-DC3, TrisQ, TMPyP4, Cu-ttpy). From the temperature dependence of the equilibrium constants, we determined the enthalpic and entropic contributions to the formation of each stoichiometric state. In absence of ligand, we untangled the potassium-induced G-quadruplex folding thermodynamics, one potassium ion at a time. The formation of each quartet-K+-quartet units is strongly enthalpy driven, with entropy penalty. In contrast, the formation of quartet-K+-triplet units is entropically driven. For this reason, such misfolded structures can become more abundant as the temperature increases. In the presence of ligands, mass spectrometry also revealed new states at intermediate temperatures. For example, even in cases where only a 1:1 (ligand:quadruplex) is observed at room temperature, a 2:1 complex predominates at intermediate temperatures. Mass spectrometry also makes it easy to distinguish ligand bound to the 2-quartet structures (containing 1 K+), the 3-quartet structures (containing 2 K+) and to the unfolded strand (no specific K+). We confirm that TrisQ binds preferably, but not exclusively, to 3-quartet structures, Phen-DC3 binds to a 2-quartet structure, while the porphyrin ligand TMPyP4 is characterized as non-selective, because it binds to all forms including the unfolded one. The thermodynamics of ligand binding to each form, one ligand at a time, provides unprecedented detail on the interplay between ligand binding and changes in G-quadruplex topology.\n\nTOC Graphics\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC=\"FIGDIR/small/370254_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@e624adorg.highwire.dtl.DTLVardef@171fa1dorg.highwire.dtl.DTLVardef@1dbc32eorg.highwire.dtl.DTLVardef@eb301f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2017

Compaction of Duplex Nucleic Acids upon Native Electrospray Mass Spectrometry

Native mass spectrometry coupled to ion mobility spectrometry is a promising tool for structural biology. Intact complexes can be transferred to the mass spectrometer and, if native conformations survive, collision cross sections give precious information on the structure of each species in solution. Based on several successful reports for proteins and their complexes, the conformation survival becomes more and more taken for granted. Here we report on the fate of nucleic acids conformation in the gas phase. Disturbingly, we found that DNA and RNA duplexes, at the electrospray charge states naturally obtained from native solution conditions ([≥] 100 mM aqueous NH4OAc), are significantly more compact in the gas phase compared to the canonical solution structures. The compaction is observed for short (12-bp) and long (36-bp) duplexes, and for DNA and RNA alike. Molecular modeling (density functional calculations on small helices, semi-empirical calculations on up to 12-bp, and molecular dynamics on up to 36-bp duplexes) demonstrates that the compaction is due to phosphate group self-solvation prevailing over Coulomb-driven expansion. Molecular dynamics simulations starting from solution structures do not reproduce the experimental compaction. To be experimentally relevant, molecular dynamics sampling should reflect the progressive structural rearrangements occurring during desolvation. For nucleic acid duplexes, the compaction observed for low charge states results from novel phosphate-phosphate hydrogen bonds formed across both grooves at the very late stages of electrospray.

biophysics↗