Unravelling the Link between Oligonucleotide Structure and Diastereomer Separation in Hydrophilic Interaction Chromatography

Analytical Chemistry, Journal Year: 2024, Volume and Issue: 96(24), P. 9994 - 10002

Published: June 10, 2024

Therapeutic oligonucleotides (ONs) commonly incorporate phosphorothioate (PS) modifications. These introduce chiral centers and generate ON diastereomers. The increasing number of ONs undergoing clinical trials reaching the market has led to a growing interest better characterize diastereomer composition, especially for small interfering ribonucleic acids (siRNAs). In this study, first time, we identify higher-order structures as major cause separation in hydrophilic interaction chromatography (HILIC). We have used conformational predictions melting profiles several representative full-length analyze folding then run mass spectrometry HILIC underpin link between their separation. On top, show how one can either enhance or suppress depending on chromatographic settings, such column temperature, pore size, stationary phase, mobile-phase ionic strength, organic modifier. This work will significantly facilitate future HILIC-based characterization PS-containing ONs; e.g., enabling monitoring batch-to-batch distributions siRNAs, complex task that is now time shown possible delicate class therapeutic double-stranded ONs.

Language: Английский

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Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

Mass Spectrometry Reviews, Journal Year: 2022, Volume and Issue: 42(5), P. 1876 - 1926

Published: June 27, 2022

Abstract Progress in structural biology research has led to a high demand for powerful and yet complementary analytical tools characterization of proteins protein complexes. This significantly increased interest native mass spectrometry (nMS), particularly top‐down (nTDMS) the past decade. review highlights recent advances nTDMS biological assemblies, with particular focus on extra multi‐layers information enabled by TDMS. We include short introduction sample preparation ionization nMS, tandem fragmentation techniques as well analyzers software/analysis pipelines used nTDMS. highlight unique offered examples its broad range applications proteins, protein‐ligand interactions (metal, cofactor/drug, DNA/RNA, protein), therapeutic antibodies antigen‐antibody complexes, membrane macromolecular machineries (ribosome, nucleosome, proteosome, viruses), endogenous The challenges, potential, along perspectives methods analysis assemblies recombinant samples are discussed.

Language: Английский

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Chloride Ligands on DNA-Stabilized Silver Nanoclusters

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 145(19), P. 10721 - 10729

Published: May 8, 2023

DNA-stabilized silver nanoclusters (AgN-DNAs) are known to have one or two DNA oligomer ligands per nanocluster. Here, we present the first evidence that AgN-DNA species can possess additional chloride lead increased stability in biologically relevant concentrations of chloride. Mass spectrometry five chromatographically isolated near-infrared (NIR)-emissive with previously reported X-ray crystal structures determines their molecular formulas be (DNA)2[Ag16Cl2]8+. Chloride exchanged for bromides, which red-shift optical spectra these emitters. Density functional theory (DFT) calculations 6-electron nanocluster show newly identified were assigned as low-occupancy silvers by crystallography. DFT also confirms crystallographic structure, yields qualitative agreement between computed and measured UV–vis absorption spectra, provides interpretation 35Cl-nuclear magnetic resonance spectrum A reanalysis structure are, fact, chlorides, yielding Using unusual (DNA)2[Ag16Cl2]8+ saline solutions a possible indicator other chloride-containing AgN-DNAs, an ligand high-throughput screening. Inclusion chlorides on AgN-DNAs presents promising new route expand diversity structure–property relationships imbue emitters favorable biophotonics applications.

Language: Английский

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Formation and Nanomechanical Properties of Silver-Mediated Guanine DNA Duplexes in Aqueous Solution

ACS Nano, Journal Year: 2024, Volume and Issue: 18(4), P. 3002 - 3010

Published: Jan. 16, 2024

Silver cations can mediate base pairing of guanine (G) DNA oligomers, yielding linear parallel G–Ag+–G duplexes with enhanced stabilities compared to those canonical duplexes. To enable their use in programmable nanotechnologies, it is critical understand solution-state formation and the nanomechanical stiffness Using temperature-controlled circular dichroism (CD) spectroscopy, we find that heating mixtures G oligomers silver salt above 50 °C fully destabilizes G-quadruplex structures converts Electrospray ionization mass spectrometry supports form at stoichiometries 1 Ag+ per pair, CD spectroscopy suggests as Ag+/base stoichiometry increases further, undergo additional morphological changes. liquid-phase atomic force microscopy, this excess enables assembly long fiberlike ∼2.5 nm heights equivalent a single duplex but lengths far exceed duplex. Finally, using conditions established duplexes, surface forces apparatus (SFA) compare solution-phase dG-dC Watson–Crick–Franklin SFA shows are 1.3 times stiffer than confirming gas-phase ion mobility measurements computational predictions. These findings may guide development structural nanotechnologies rely on silver-mediated pairing.

Language: Английский

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Accessing Different Protein Conformer Ensembles with Tunable Capillary Vibrating Sharp-Edge Spray Ionization

The Journal of Physical Chemistry B, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 29, 2025

Capillary vibrating sharp-edge spray ionization (cVSSI) has been used to control the droplet charging of nebulized microdroplets and monitor effects on protein ion conformation makeup as determined by mass spectrometry (MS). Here it is observed that application voltage results in noticeable differences charge state distributions (CSDs) ubiquitin ions. The data can be described most generally three distinct regions: Under low-voltage conditions (<+200 V, LV regime), low states (2+ 4+ ions) dominate spectra. For midvoltage (+200 +600 MV higher (7+ 12+ are observed. high-voltage (>+600 HV "nano-electrospray (nESI)-type distribution" achieved which 6+ 5+ species dominant Analysis these suggests different pathways progeny nanodroplet production result regime, aerodynamic breakup leads droplets selective for native solution ensemble (minus multimeric species). In large persist longer periods time, leading heating a shift partially unfolded species. access nanodroplets faster, sampling indicated nESI-type CSD. notable observation limited multimer formation adduct regime hypothesized from aero resulting carrier partitioning sampled droplets. tunable afforded cVSSI presents opportunities study charge, size, spectrometer inlet temperature conformer spectrometer. Additionally, approach may provide tool rapid comparison stabilities.

Language: Английский

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Structural insights and shedding light on preferential interactions of dietary flavonoids with G-quadruplex DNA structures: A new horizon

Heliyon, Journal Year: 2023, Volume and Issue: 9(3), P. e13959 - e13959

Published: Feb. 20, 2023

G-quadruplex, a structurally unique structure in nucleic acids present all throughout the human genome, has sparked great attention therapeutic investigations. Targeting G-quadruplex is new strategy for drug development. Flavonoids are found almost dietary plant-based beverages and food products; therefore, they ingested significant proportions through diet. Although synthetically developed molecules used vigorously but have various adverse effects. While on other hand, nature supplies chemically scaffolds form of distinct flavonoids that easily accessible, less poisonous, higher bioavailability. Because their pharmacological effectiveness minimal cytotoxicity, such low molecular weight compounds feasible alternatives to synthetic medicines. Therefore, from drug-development point view, investigation screening binding capabilities quadruplex-interactive small natural like expected be highly effective, with particular emphasis selectivity towards polymorphic structures. In this respect, quadruplexes scintillated research into potential interaction these flavonoids. The purpose review offer an up-to-date close-up look at varied goal providing newer perspectives construct novel agents next-generation disease managements.

Language: Английский

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Collision-Induced Unfolding Reveals Disease-Associated Stability Shifts in Mitochondrial Transfer Ribonucleic Acids

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: 146(7), P. 4412 - 4420

Published: Feb. 8, 2024

Ribonucleic acids (RNAs) remain challenging targets for structural biology, creating barriers to understanding their vast functions in cellular biology and fully realizing applications biotechnology. The inherent dynamism of RNAs creates numerous obstacles capturing biologically relevant higher-order structures (HOSs), as a result, many RNA unknown. In this study, we describe the development native ion mobility-mass spectrometry collision-induced unfolding (CIU) characterization variety RNAs. We evaluate ability these techniques preserve features gas phase across wide range functional Finally, apply tools study elusive mitochondrial encephalopathy, lactic acidosis, stroke-like episodes-associated A3243G mutation. Our data demonstrate that our experimentally determined conditions some solution-state memory via correlated complexity CIU fingerprints HOS, observation predicted stability shifts control samples, retention magnesium binding events gas-phase ions. Significant differences collision cross section are observed function mutation subset tRNA maturation pathway. conclude by discussing potential application RNA-based biotherapeutics and, more broadly, transcriptomic characterization.

Language: Английский

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Effects of Nano-Electrospray Ionization Emitter Position on Unintentional In-Source Activation of Peptide and Protein Ions

Journal of the American Society for Mass Spectrometry, Journal Year: 2024, Volume and Issue: 35(3), P. 498 - 507

Published: Feb. 20, 2024

Native ion mobility-mass spectrometry (IM-MS) typically introduces protein ions into the gas phase through nano-electrospray ionization (nESI). Many nESI setups have mobile stages for tuning signal and extent of co-solute salt adduction. However, position emitter capillary in can unintended downstream consequences collision-induced unfolding or dissociation (CIU/D) experiments. Here, we show that relatively small variations shift midpoint (commonly called "CID50" "CIU50") potential CID breakdown curves CIU transitions by as much 8 V on commercial instruments. A spatial "map" CID50 loss heme from holomyoglobin onto a Waters Synapt G2-S

Language: Английский

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Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry

Molecular & Cellular Proteomics, Journal Year: 2024, Volume and Issue: 23(4), P. 100742 - 100742

Published: Feb. 23, 2024

Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates with a sulfur atom place of standard nonbridging oxygen, is the most common modifications because ease pharmacokinetic benefits. Quality assessment RNA synthesis, including modification incorporation, essential for selectivity performance, synthetic nature PS linkage incorporation often reveals impurities. Here, we present comprehensive analysis via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS especially useful diagnosing producing diagnostic a- z-type ions at sites, beyond d- w-type ions. Analysis using resonant beam-type collision-based activation that, overall, intense sequence base-loss result when present. Furthermore, report increased detection b- x-type product sites addition c- y-type This work gas-phase chemical stability afforded by alters necessitates inclusion additional RNA.

Language: Английский

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DNA Stabilizes Eight-Electron Superatom Silver Nanoclusters with Broadband Downconversion and Microsecond-Lived Luminescence

The Journal of Physical Chemistry Letters, Journal Year: 2022, Volume and Issue: 13(35), P. 8305 - 8311

Published: Aug. 29, 2022

DNA oligomers are known to serve as stabilizing ligands for silver nanoclusters (AgN-DNAs) with rod-like nanocluster geometries and nanosecond-lived fluorescence. Here, we report two AgN-DNAs that possess distinctly different structural properties the first exhibit only microsecond-lived luminescence. These emitters characterized by significant broadband downconversion from ultraviolet/visible near-infrared region. Circular dichroism spectroscopy shows structures of these differ significantly previously reported AgN-DNAs. We find contain eight valence electrons, making them DNA-stabilized luminescent quasi-spherical superatoms. This work demonstrates important role composition geometry play in dictating luminescence expands space structure–property relations can be achieved

Language: Английский

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