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See detailRecognition of homopyrimidine mismatches by distance-constrained macrocyclic bisintercalators
Bahr, Matthias; Gabelica, Valérie ULg; Granzhan, Anton et al

in Nucleic Acids Symposium Series (2008), 52

Binding of three macrocyclic bisintercalators to mismatch-containing duplexes was analyzed by thermal denaturation experiments, electrospray mass spectrometry studies (ESI-MS) and fluorescent intercalator ... [more ▼]

Binding of three macrocyclic bisintercalators to mismatch-containing duplexes was analyzed by thermal denaturation experiments, electrospray mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bisintercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched one and affinity in the submicromolar range (Kd). The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine-pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch [less ▲]

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See detailTop-Down Proteomics using Matrix-Enhanced ISD
Demeure, Kevin ULg; Quinton, Loïc ULg; Gabelica, Valérie ULg et al

Poster (2007, October 11)

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See detailExploring the Formation Pathways of DNA G-Quadruplex Architectures
Rosu, Frédéric ULg; Poncelet, Harmonie; Teulade-Fichou, Marie-Paule et al

Conference (2007, September 07)

Guanine-rich DNA strands can form the so-called G-quadruplex architectures due to the formation of quartets of guanines linked by 8 hydrogen bonds. G-quadruplexes are further stabilized by the inclusion ... [more ▼]

Guanine-rich DNA strands can form the so-called G-quadruplex architectures due to the formation of quartets of guanines linked by 8 hydrogen bonds. G-quadruplexes are further stabilized by the inclusion of cations between the G-quartets. The abundance of G-rich regions throughout the genome and their very presence in telomeric regions made G-quadruplexes interesting targets. NMR and crystallographic studies of G-quadruplex structures revealed amazing variety in the G-quadruplex topologies. The next challenge will be to understand the rules governing the formation of the various topologies, in order to predict relevant G-quadruplexes in the genome, and in order to act rationally on their formation or disruption. To date, only few experimental [1] or theoretical [2] studies have been devoted to investigating the mechanisms of G-quadruplex formation. We report here a detailed investigation of DNA G-quadruplex formation pathways using electrospray mass spectrometry (ESI-MS). The sequences TGnT (n = 3-6) were purchased from Eurogentec (Seraing, Beliugm). ESI-MS experiments were performed in the negative ion mode on a Q-TOF Ultima Global (Waters, Manchester, UK). The cation used was ammonium (up to 150 mM). Experiments were performed in the presence and absence of methanol (up to 20%) as co-solvent. ESI-MS allows counting both the number of strands and the number of cations in each intermediate. We could confirm the presence of transient dimer and trimer intermediates in low abundance. More unexpectedly, ESI-MS also reveals unambiguously the formation of pentamers which contain ammonium cations. The pentamers slowly convert into tetramers. Counting the number of included cations also revealed that, in the case of (TG6T)4, inclusion of four ammonium cations is fast, while the inclusion of the last ammonium ion is very slow. We also found that the addition of methanol (initially added to obtain higher ion intensities) significantly increases the rate of G-quadruplex formation. Finally, we also investigated the role of G-quadruplex ligands in the rate of formation of G-quadruplexes. We could classify the ligands according to their increase of G-quadruplex formation kinetics, and distinguish the intermediates. Interestingly, one ligand showed formation of a higher-order structure by bridging two G-quadruplexes. Acknowledgement: The authors thank the FRS-FNRS for their support. References: [1] J. Gros et al., Nucleic Acids Res., 2007, doi:10.1093/nar/gkm111. [2] R. Stefl et al., Biophys. J., 2003, 85(3), 1787-1804. [less ▲]

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See detailTop-Down Proteomics using Matrix-Enhanced ISD
Demeure, Kevin ULg; Quinton, Loïc ULg; Gabelica, Valérie ULg et al

Conference (2007, June 05)

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See detailPhotodetachment and Photodissociation Mass Spectrometry of Biomolecule Ions
Gabelica, Valérie ULg

Conference (2007, April 27)

We recently explored the effects of irradiating DNA polyanions stored in a quadrupole ion trap (QIT) mass spectrometer with an optical parametric oscillator (OPO) laser between 250 nm and 285 nm. We ... [more ▼]

We recently explored the effects of irradiating DNA polyanions stored in a quadrupole ion trap (QIT) mass spectrometer with an optical parametric oscillator (OPO) laser between 250 nm and 285 nm. We studied DNA 6-mer to 20-mer single strands, and 12-base pair double strands. In all cases, laser irradiation causes electron detachment from the multiply charged DNA anions. Electron photo-detachment efficiency directly depends on the number of guanines in the strand, and maximum efficiency is observed between 260 and 275 nm. Collisional activation of the radical anions results in extensive fragmentation, which can be used to sequence the DNA strands. It has therefore important potential applications in analytical chemistry. We also obtained preliminary results on laser irradiation of DNA coupled to other chromophores (covalently bound or noncovalently bound). Depending on the chromophore, three different behaviors are encountered: (1) the photon energy can be redistributed in the molecule by internal conversion, (2) electron photodetachment was observed for a few chromophores, and (3) specific photodissociation was observed for a porphyrin chromophore. Here we will address the question of the mechanism of electron photodetachment. DNA multiply charged anions undergo one-photon electron ejection (oxidation) when subjected to laser irradiation at 260 nm (4.77 eV). Electron photodetachment is likely a fast process, given that photodetachment is able to compete with internal conversion and/or radiative relaxation to the ground state. The DNA [6-mer]3- ions studied here show a marked sequence-dependence of electron photodetachment yield. Remarkably, the photodetachment yield (dG6 > dA6 > dC6 > dT6) is inversely correlated with the base ionization potentials (G < A < C < T). Sequences with guanine runs show increased photodetachment yield as the number of guanine increases, in line with the fact that positive holes are the most stable in guanine runs. This correlation between photodetachment yield and the stability of the base radical may be explained by tunneling of the electron through the repulsive Coulomb barrier. The wavelength-dependence of electron detachment yield was studied for dG63-. Maximum electron photodetachment is observed in the wavelength range corresponding to base absorption (260-270 nm). This demonstrates the feasibility of gas-phase UV spectroscopy on large DNA anions. The calculations and the wavelength dependence suggest that the electron photodetachment is initiated at the bases and not at the phosphates. This also indicates that, although direct photodetachment could also occur, autodetachment from excited states, presumably corresponding to base excitation, is the dominant process at 260 nm. Excited states dynamics of large DNA strands still remains largely unexplored, and photo-oxidation studies on trapped DNA multiply charged anions can help bridging the gap between gas-phase studies on isolated bases or base pairs and solution-phase studies on full DNA strands. Time-resolved measurements on photodetachment and photodissociation can be used to contribute elucidating the mechanisms at stake in these novel experiments. [less ▲]

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See detailWhen Mass Spectrometry meets Spectroscopy: A Different Perspective on Ion Photodissociation
Gabelica, Valérie ULg

Conference (2007, February 27)

Photodissociation methods are more and more widely used in analytical mass spectrometry as an alternative to collision-induced dissociation. A well known example is infrared multiphoton dissociation ... [more ▼]

Photodissociation methods are more and more widely used in analytical mass spectrometry as an alternative to collision-induced dissociation. A well known example is infrared multiphoton dissociation (IRMPD) with CO2 lasers (wavelength 10.6 µm), which is often preferred to SORI-CID in FTICR mass spectrometers. IR photons are absorbed by the vibrational modes of the ion, and this vibrational energy is redistributed all over the molecule. The fragmentation channels observed are usually the same as for CID. Recently, new possibilities offered by UV lasers of fixed wavelength (i.e. 157 nm or 193 nm) for peptide fragmentation were described. On the contrary to IRMPD, irradiation at these UV wavelengths gives rise to fragmentation channels which are unprecedented in CID, suggesting that photochemical reactions take place. The prerequisite for ion photodissociation is photon absorption. In the infrared, photon absorption results in the excitation of vibrational modes. Anharmonic coupling of the excited vibrational modes with other vibrational modes of the ion results in a rapid intramolecular vibrational energy redistribution (IVR). The energy of an IR photon is too low for the ion to dissociate (0.117 eV/photon for a 10.6 µm CO2 laser). Multiple cycles of absorption and IVR are necessary for IR photodissociation to occur (hence the term multiphoton). Because the first step in photodissociation is photon absorption, the recording of the photodissociation yield as a function of the photon wavenumber gives information on the vibrational modes of the ion. This is the basic principle of infrared spectroscopy experiments inside the mass spectrometer. This technique will be illustrated by a recent example on DNA single strands and quadruplex structures. The shifting of the vibrational frequencies corresponding to guanine C=O stretch gives information on the base pairing in the DNA ion in the gas phase. The situation is somewhat different with UV photons, which may lead to either electronic excitation of the ions or direct electron ejection. Electronic excited states may evolve in different ways: (1) Internal conversion to a vibrationally excited electronic ground state. Resulting fragments would then be similar to CID and IRMPD. This process can be multiphotonic and could be coined UVMPD. (2) Electron autodetachment, leading to the formation of radicals that can be further fragmented to obtain ECD-like or EDD-like fragments. (3) Photochemical reactions giving rise to unprecedented fragmentation channels. In all three cases, product ion yield is conditioned by photon absorption by an electronic state, meaning that UV spectroscopy of gas-phase ions can be performed with a mass spectrometer. We recently explored the effects of photon absorption on multiply charged DNA anions. Depending on the photon wavelength and on the chromophores, all three situations could be observed. Electron photodetachment is the major channel observed when using wavelengths corresponding to DNA base absorption.1,2 When irradiated at proper wavelength, chromophoric DNA ligands also give rise to electron photodetachment. UV-visible spectra of the DNA duplexes and complexes were recorded and compared with solution-phase spectra. Finally, a porphyrin ligand gives rise to specific fragmentation pathways when irradiated at 450 nm, while photodetachment is observed at 260 nm. The most recent results will be presented and discussed. 1. Gabelica, V.; Tabarin, T.; Antoine, R.; Rosu, F.; Compagnon, I.; Broyer, M.; De Pauw, E.; Dugourd, P. Electron Photodetachment Dissociation of DNA Polyanions in a Quadrupole Ion Trap Mass Spectrometer. Anal. Chem. 2006, 78 (18), 6564. 2. Gabelica, V.; Rosu, F.; Tabarin, T.; Kinet, C.; Antoine, R.; Broyer, M.; De Pauw, E.; Dugourd, P. Base-Dependent Electron Photodetachment from Negatively Charged DNA Strands upon 260-nm Laser Irradiation. J. Am. Chem. Soc. 2007, accepted for publication. [less ▲]

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See detailRational selection of the optimum MALDI matrix for top-down proteomics by in-source decay
Demeure, Kevin ULg; Quinton, Loïc ULg; Gabelica, Valérie ULg et al

in Analytical Chemistry (2007), 79(22), 8678-8685

in-source decay (ISD) in MALDI leads to c- and z-fragment ion series enhanced by hydrogen radical donors and is a useful method for sequencing purified peptides and proteins. Until now, most efforts to ... [more ▼]

in-source decay (ISD) in MALDI leads to c- and z-fragment ion series enhanced by hydrogen radical donors and is a useful method for sequencing purified peptides and proteins. Until now, most efforts to improve methods using ISD concerned instrumental optimization. The most widely used ISD matrix is 2,5-dihydroxybenzoic acid (DHB). We present here a rational way to select MALDI matrixes likely to enhance ISD for top-down proteomic approaches. Starting from Takayama's model (Takayama, M. J. Am. Soc. Mass Spectrom. 2001, 12, 1044-9), according to which formation of ISD fragments (c and z) would be due to a transfer of hydrogen radical from the matrix to the analyte, we evaluated the hydrogen-donating capacities of matrixes, and thus their ISD abilities, with spirooxazines (hydrogen scavengers). The determined hydrogen-donating abilities of the matrixes are ranked as follows: picolinic acid (PA) > 1,5-diaminonaphtalene (1,5-DAN) > DHB > sinapinic acid > alpha-cyano-4-hydroxycinnamic acid. The ISD enhancement obtained by using 1,5-DAN compared to DHB was confirmed with peptides and proteins. On that basis, a matrix-enhanced ISD approach was successfully applied to sequence peptides and proteins up to similar to 8 kDa. Although PA alone is not suitable for peptide and protein ionization, ISD signals could be further enhanced when PA was used as an additive to 1,5-DAN. The optimized matrix preparation was successfully applied to identify larger proteins by large ISD tag researches in protein databases (BLASTp). Coupled with an adequate separation method, ISD is a promising tool to include in a top-down proteomic strategy. [less ▲]

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See detailStabilization and structure of telomeric and c-myc region intramolecular G-quadruplexes: The role of central cations and small planar ligands
Gabelica, Valérie ULg; Baker, Erin Shammel; Teulade-Fichou, Marie-Paule et al

in Journal of the American Chemical Society (2007), 129(4), 895-904

A promising approach for anticancer strategies is the stabilization of telomeric DNA into a G-quadruplex structure. To explore the intrinsic stabilization of folded G-quadruplexes, we combined ... [more ▼]

A promising approach for anticancer strategies is the stabilization of telomeric DNA into a G-quadruplex structure. To explore the intrinsic stabilization of folded G-quadruplexes, we combined electrospray ionization mass spectrometry, ion mobility spectrometry, and molecular modeling studies to study different DNA sequences known to form quadruplexes. Two telomeric DNA sequences of different lengths and two DNA sequences derived from the NHE III1 region of the c-myc oncogene (Pu22 and Pu27) were studied. NH4+ and the ligands PIPER, TMPyP4, and the three quinacridines MMQ1, MMQ3, and BOQ1 were complexed with the DNA sequences to determine their effect on the stability of the G-quadruplexes. Our results demonstrate that G-quadruplex intramolecular folds are stabilized by NH4+ cations and the ligands listed. Furthermore, the ligands can be classified according to their ability to stabilize the quadruplexes and end stacking is shown to be the dominant mode for ligand attachment. In all cases our solvent-free experimental observations and theoretical modeling reveal structures that are highly relevant to the solution-phase structures. [less ▲]

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See detailGuanines are a quartet's best friend: impact of base substitutions on the kinetics and stability of tetramolecular quadruplexes
Gros, Julien; Rosu, Frédéric ULg; Amrane, Samir et al

in Nucleic Acids Research (2007), 35(9), 3064-3075

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in ... [more ▼]

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in which each guanine of the central block was systematically substituted with a different base. Twelve types of substitutions were assessed in more than 100 different sequences. We conducted a comparative kinetic analysis of all tetramers. Electrospray mass spectrometry was used to count the number of inner cations, which is an indicator of the number of effective tetrads. In general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, resulting in reduced quadruplex lifetime/ thermal stability and in decreased association rate constants. We demonstrate extremely large differences in the association rate constants of these quadruplexes depending on modification position and type. These results demonstrate that most guanine substitutions are deleterious to tetramolecular quadruplex structure. Despite the presence of well- defined non- guanine base quartets in a number of NMR and X- ray structures, our data suggest that most non- guanine quartets do not participate favorably in structural stability, and that these quartets are formed only by virtue of the docking platform provided by neighboring G- quartets. Two notable exceptions were found with 8- bromoguanine ( X) and 6- methyl- isoxanthopterin ( P) substitutions, which accelerate quadruplex formation by a factor of 10 when present at the 50 end. The thermodynamic and kinetic data compiled here are highly valuable for the design of DNA quadruplex assemblies with tunable association/ dissociation properties. [less ▲]

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See detailBase-dependent electron photodetachment from negatively charged DNA strands upon 260-nm laser irradiation
Gabelica, Valérie ULg; Rosu, Frédéric ULg; Tabarin, Thibault et al

in Journal of the American Chemical Society (2007), 129(15), 4706-4713

DNA multiply charged anions stored in a quadrupole ion trap undergo one-photon electron ejection (oxidation) when subjected to laser irradiation at 260 nm (4.77 eV). Electron photodetachment is likely a ... [more ▼]

DNA multiply charged anions stored in a quadrupole ion trap undergo one-photon electron ejection (oxidation) when subjected to laser irradiation at 260 nm (4.77 eV). Electron photodetachment is likely a fast process, given that photodetachment is able to compete with internal conversion or radiative relaxation to the ground state. The DNA [6-mer](3-) ions studied here show a marked sequence dependence of electron photodetachment yield. Remarkably, the photodetachment yield (dG(6) > dA(6) > dC(6) > dT(6)) is inversely correlated with the base ionization potentials (G < A < C < T). Sequences with guanine runs show increased photodetachment yield as the number of guanine increases, in line with the fact that positive holes are the most stable in guanine runs. This correlation between photodetachment yield and the stability of the base radical may be explained by tunneling of the electron through the repulsive Coulomb barrier. Theoretical calculations on dinucleotide monophosphates show that the HOMO and HOMO-1 orbitals are localized on the bases. The wavelength dependence of electron detachment yield was studied for dG(6)(3-). Maximum electron photodetachment is observed in the wavelength range corresponding to base absorption (260-270 nm). This demonstrates the feasibility of gas-phase UV spectroscopy on large DNA anions. The calculations and the wavelength dependence suggest that the electron photodetachment is initiated at the bases and not at the phosphates. This also indicates that, although direct photodetachment could also occur, autodetachment from excited states, presumably corresponding to base excitation, is the dominant process at 260 nm. Excited-state dynamics of large DNA strands still remains largely unexplored, and photo-oxidation studies on trapped DNA multiply charged anions can help in bridging the gap between gas-phase studies on isolated bases or base pairs and solution-phase studies on full DNA strands. [less ▲]

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See detailElectron photodetachment dissociation of DNA anions with covalently or noncovalently bound chromophores
Gabelica, Valérie ULg; Rosu, Frédéric ULg; De Pauw, Edwin ULg et al

in Journal of the American Society for Mass Spectrometry (2007), 18(11), 1990-2000

Double stranded DNA multiply charged anions coupled to chromophores were subjected to UV-Vis photoactivation. in a quadrupole ion trap mass spectrometer. The chromophores included noncovalently bound ... [more ▼]

Double stranded DNA multiply charged anions coupled to chromophores were subjected to UV-Vis photoactivation. in a quadrupole ion trap mass spectrometer. The chromophores included noncovalently bound minor groove binders (activated in the near UV), noncovalently bound intercalators (activated with visible light), and covalently linked fluorophores and quenchers (activated at their maximum absorption wavelength). We found that the activation of only chromophores having long fluorescence lifetimes did result in efficient electron photodetachment from the DNA complexes. In the case of ethidium-dsDNA complex excited at 500 nm, photodetachment is a multiphoton process. The MS3 fragmentation of radicals produced by photodetachment at lambda = 260 nm (DNA excitation) and by photodetachment at lambda > 300 nm (chromophore excitation) were compared. The radicals keep no memory of the way they were produced. A weakly bound noncovalent ligand (m-amsacrine) allowed probing experimentally that a fraction of the electronic internal energy was converted into vibrational internal energy. This fragmentation channel was used to demonstrate that excitation of the quencher DABSYL resulted in internal conversion, unlike the fluorophore 6-FAM. Altogether, photodetachment of the DNA complexes upon chromophore excitation can be interpreted by the following mechanism: (1) ligands with sufficiently long excited-state lifetime undergo resonant two-photon excitation to reach the level of the DNA excited states, then (2) the excited-state must be coupled to the DNA excited states for photodetachment to occur. Our experiments also pave the way towards photodissociation probes of biomolecule conformation in the gas-phase by Forster resonance energy transfer (FRET). [less ▲]

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See detailLigand binding mode to duplex and triplex DNA assessed by combining electrospray tandem mass spectrometry and molecular modeling
Rosu, Frédéric ULg; Nguyen, Chi-Hung; De Pauw, Edwin ULg et al

in Journal of the American Society for Mass Spectrometry (2007), 18(6), 1052-1062

In this paper, we report the analysis of seven benzopyridoindole and benzopyridoquinoxaline drugs binding to different duplex DNA and triple helical DNA, using an approach combining electrospray ... [more ▼]

In this paper, we report the analysis of seven benzopyridoindole and benzopyridoquinoxaline drugs binding to different duplex DNA and triple helical DNA, using an approach combining electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS/MS), and molecular modeling. The ligands were ranked according to the collision energy (CE(50)) necessary to dissociate 50% of the complex with the duplex or the triplex in tandem MS. To determine the probable ligand binding site and binding mode, molecular modeling was used to calculate relative ligand binding energies in different binding sites and binding modes. For duplex DNA binding, the ligand-DNA interaction energies are roughly correlated with the experimental CE(50), with the two benzopyridoindole ligands more tightly bound than the benzopyridoquinoxaline ligands. There is, however, no marked AT versus GC base preference in binding, as supported both by the ESI-MS and the calculated ligand binding energies. Product ion spectra of the complexes with triplex DNA show only loss of neutral ligand for the benzopyridoquinoxalines, and loss of the third strand for the benzopyridoindoles, the ligand remaining on the duplex part. This indicates a higher binding energy of the benzopyridoindoles, and also shows that the ligands interact with the triplex via the duplex. The ranking of the ligand interaction energies compared with the CE(50) values obtained by MS/MS on the complexes with the triplex clearly indicates that the ligands intercalate via the minor groove of the Watson-Crick duplex. Regarding triplex versus duplex selectivity, our experiments have demonstrated that the most selective drugs for triplex share the same heteroaromatic core. [less ▲]

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See detailFormation de radicaux anions d'ADN par irradiation laser UV et visible et nouveaux canaux de dissociation
Gabelica, Valérie ULg; Tabarin, Thibault; Kinet, Catherine ULg et al

Conference (2006, September 13)

Nous avons exploré récemment les effets de l'irradiation laser dans l'UV ou le visible de polyanions d'ADN dans un piège à ions quadripolaire. Lors de l'irradiation de simple brins ou de double hélices d ... [more ▼]

Nous avons exploré récemment les effets de l'irradiation laser dans l'UV ou le visible de polyanions d'ADN dans un piège à ions quadripolaire. Lors de l'irradiation de simple brins ou de double hélices d'ADN en UV autour de 260 nm, nous avons observé un détachement d'électrons, donnant lieu à des radicaux anions. L'étude de la dépendence de l'efficacité de ce photodétachement d'électrons en fonction de la séquence des brins a révélé que ce phénomène était corrélé au potentiel d'ionisation des bases nucléiques. La guanine est la base la plus facilement oxydée, suivie de l'adénine, de la cytosine et de la thymine. En pratique, plus un brin contient de guanines, plus la formation de radicaux par photodétachement d'électrons est efficace. L'efficacité du photodétachement montre également une dépendence envers l'état de charge du brin (les plus chargés ont une efficacité de détachement plus élevée) et en fonction de la longueur d'onde (avec un maximum d'efficacité autour de 265 nm), montrant que la transition vers un état électronique excité des bases facilite le détachement d'électrons. Plus surprenant encore, des complexes avec des chromophores (ligands du sillon de l'ADN et intercalants) montrent également un photodétachement d'électrons, et ce avec des énergies de photons encore plus basses, correspondant à une absorption dans le visible (i.e. 550 nm pour l'éthidium). Ceci montre que les énergies de liaisons des électrons sont relativement faibles, puisqu'ils peuvent être détachés par des photons de 2.25 eV. Nous avons également étudié la fragmentation des radiaux ainsi produits, par CID dans le piège. Dans le cas des complexes avec les chromophores, les canaux de fragmentation principaux sont des pertes de neutres, y compris des morceaux de ligands. Il est intéressant de constater que les radicaux se fragmentent de la même manière, quel que soit le chromophore irradié qui a donné lieu à sa formation. Cecis suggère que les radicaux se réorganisent rapidement par rapport au temps de l'expérience. Dans le cas de simples brins (12-mères à 20-mères), nous avons constaté une fragmentation plus efficace des radicaux comparés aux ions à couches complètes de même charge. Plus intéressant, les canaux de dissociation sont très particuliers : alors que la CID sur ions à couches complètes donne majoritairement des ions 'w' et 'a-Base', la CID sur les radicaux donne des ions 'w', 'd', 'a radicalaires', et 'z radicalaires'. Il est aisé d'obtenir une bonne couverture de séquence à des énergies de collision faibles. A nouveau, bien que le détachement d'électrons soit initié par les bases guanines, la couverture de séquence est indépendante de la composition, suggérant une redistribution dans les radicaux. L'avantage principal de la fragmentation des radicaux est l'absence de fragments internes (ou 'double fragments'), ce qui facilite le séquencage de novo. De plus, selon notre expérience, la formation des radicaux est plus aisée par irradiation laser que par EDD. [less ▲]

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See detailElectron photodetachment dissociation of DNA polyanions in a quadrupole ion trap mass spectrometer
Gabelica, Valérie ULg; Tabarin, Thibault; Antoine, Rodolphe et al

in Analytical Chemistry (2006), 78(18), 6564-6572

We hereby explore the effects of irradiating DNA polyanions stored in a quadrupole ion trap mass spectrometer with an optical parametric oscillator laser between 250 and 285 nm. We studied DNA 6-20-mer ... [more ▼]

We hereby explore the effects of irradiating DNA polyanions stored in a quadrupole ion trap mass spectrometer with an optical parametric oscillator laser between 250 and 285 nm. We studied DNA 6-20-mer single strands and 12-base pair double strands. In all cases, laser irradiation causes electron detachment from the multiply charged DNA anions. Electron photodetachment efficiency directly depends on the number of guanines in the strand, and maximum efficiency is observed between 260 and 275 nm. Subsequent collision-induced dissociation (CID) of the radical anions produced by electron photodetachment results in extensive fragmentation. In addition to neutral losses, a large number of fragments from the w, d, a*, and z* ion series are obtained, contrasting with the w and (a-base) ion series observed in regular CID. The major advantage of this technique, coined electron photodetachment dissociation (EPD) is the absence of internal fragments, combined with good sequence coverage. EPD is therefore a highly promising approach for de novo sequencing of oligonucleotides. EPD of nucleic acids is also expected to give specific radical-induced strand cleavages, with conservation of other fragile bonds, including noncovalent bonds. In effect, preliminary results on a DNA hairpin and on double strands suggest that EPD could also be used to probe intra- and intermolecular interactions in nucleic acids. [less ▲]

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See detailPositive and negative ion mode ESI-MS and MS/MS for studying drug-DNA complexes
Rosu, Frédéric ULg; Pirotte, Sophie ULg; De Pauw, Edwin ULg et al

in International Journal of Mass Spectrometry (2006), 253(3), 156-171

We report systematic investigation of duplex DNA complexes with minor groove binders (Hoechsts 33258 and 33342, netropsin and DAPI) and intercalators (daunomycin, doxorubicin, actinomycin D, ethidium ... [more ▼]

We report systematic investigation of duplex DNA complexes with minor groove binders (Hoechsts 33258 and 33342, netropsin and DAPI) and intercalators (daunomycin, doxorubicin, actinomycin D, ethidium, cryptolepine, neocryptolepine, m-Amsacrine, proflavine, ellipticine and mitoxantrone) by ESI-MS and ESI-MS/MS in the negative ion mode and in the positive ion mode. The apparent solution phase equilibrium binding constants can be determined by measuring relative intensities in the ESI-MS spectrum. While negative ion mode gives reliable results, positive ion mode gives a systematic underestimation of the binding constants and even a complete suppression of the complexes for intercalators lacking functional groups capable of interacting in the grooves. In the second part of the paper we systematically compare MS/MS fragmentation channels and breakdown curves in the positive and the negative modes, and discuss the possible uses and caveats of MS/MS in drug-DNA complexes. In the negative mode, the drugs can be separated in three groups: (1) those that leave the complex with no net charge; (2) those that leave the complex with a negative charge; and (3) those that remain attached on the strands upon dissociation of the duplex due to their positive charge. In the positive ion mode, all complexes fragment via the loss of protonated drug. Information on the stabilization of the complex by drug-DNA noncovalent interactions can be obtained straightforwardly only in the case of neutral drug loss. In all other cases, proton affinity (in the positive ion mode), gas-phase basicity (in the negative ion mode) and coulombic repulsion are the major factors influencing the fragmentation channel and the dissociation kinetics. [less ▲]

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See detailG-quadruplexes in telomeric repeats are conserved in a solvent-free environment
Baker, Erin Shammel; Bernstein, Summer L; Gabelica, Valérie ULg et al

in International Journal of Mass Spectrometry (2006), 253(3), 225-237

The structural properties of G-quadruplex forming sequences. such as the human telomeric repeat d(T(2)AG(3))(n), are of great interest due to their role in cancer and cellular aging. To determine if G ... [more ▼]

The structural properties of G-quadruplex forming sequences. such as the human telomeric repeat d(T(2)AG(3))(n), are of great interest due to their role in cancer and cellular aging. To determine if G-quadruplexes are present in a solvent-free environment. different lengths of the telomeric repeat d(T(2)AG(3))(n) (where n = 1, 2, 4 and 6) and dTG(4)T were investigated with mass specteometry, ion mobility and molecular dynamics calculations. Nano-ESI-MS illustrated quadruples stoichiometries compatible with G-quadruplex Structure.; for each sequence, with dT(2)AG(3) and dTG(4)T forming 4-strand complexes with two and three NH4+ adducts. d(T(2)AG(3))(2) a 2-strand complex. and d(T(2)AG(3))(4) and d(T(2)AG(3))(6) remaining single-stranded. Experimental cross sections were obtained for all species using ion mobility method.,. In all case.,. these could be quantitatively matched to model cross sections with specific strand orientations (parallel/antiparallel) and structures. For each species, the solvent-free structures agreed with the solution CD measurements, but the ion mobility/modeling procedure often gave Much more detailed structural information. (C) 2006 Elsevier B.V. All rights reserved. [less ▲]

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