Ligands playing musical chairs with G-quadruplex DNA: a rapid and simple displacement assay for identifying selective G-quadruplex binders.

We report here the details of G4-FID (G-quadruplex fluorescent intercalator displacement), a simple method aiming at evaluating quadruplex-DNA binding affinity and quadruplex- over duplex-DNA selectivity of putative ligands. This assay is based on the loss of fluorescence upon displacement of thiazole orange from quadruplex- and duplex-DNA matrices. The original protocol was tested using various quadruplex- and duplex-DNA targets, and with a wide panel of G-quadruplex ligands belonging to different families (i.e. from quinacridines to metallo-organic ligands) likely to display various binding modes. The reliability of the assay is further supported by comparisons with FRET-melting and ESI-MS assays.

Influence of the matrix on analyte fragmentation in atmospheric pressure MALDI.

In this paper, we report the measurement of the degree of analyte fragmentation in AP-MALDI as a function of the matrix and of the laser fluence. The analytes include p-OCH3-benzylpyridinium, three peptides containing the sequence EEPP (which cleave very efficiently at the E-P site), and three deoxynucleosides (dA, dG, and dC), which lose the neutral sugar to give the protonated base. We found that the matrix hardness/softness was consistent when comparing the analytes, with a consensus ranking from hardest to softest: CHCA >> DHB > SA approximately THAP > ATT > HPA. However, the exact ranking can be fluence-dependent, for example between ATT and HPA. Our goal here was to provide the scientific community with a detailed dataset that can be used to compare with theoretical predictions. We tried to correlate the consensus ranking with different matrix properties: sublimation or decomposition temperature (determined using thermogravimetry), analyte initial velocity, and matrix proton affinity. The best correlation was found with the matrix proton affinity.

Thermometer ions for matrix-enhanced laser desorption/ionization internal energy calibration.

This work describes a method to use relative fragmentation yields to characterize the internal energy distribution of ions produced by matrix-enhanced laser desorption/ionization mass spectrometry (MELDI-MS, see: Wright LG, Cooks RG, Wood KL. Biomed. Mass Spectrom. 1985; 12: 153-162). Assuming that the fragmentation proceeds statistically and that the collisions in the source lead to a Boltzmann-like distribution of the internal energy, a characteristic parameter, the effective temperature, is introduced to describe the internal energy distribution of the ions observed. The hypotheses, advantages and drawbacks of the implementation of the method that uses substituted benzylpyridinium salts as thermometer ions are discussed. Use is made of two matrices that produce no matrix cations in MELDI and are suitable for small cationic salts. The actual value of this effective temperature significantly depends on an accurate determination of the threshold dissociation energies and on the time spent in the source, in addition to the statistical hypothesis itself. The method could be applied to normalize spectra in order to compare results issued from different instruments.

Comparison between solution-phase stability and gas-phase kinetic stability of oligodeoxynucleotide duplexes.

The relative kinetic stabilities of different 16-mer oligonucleotide duplexes were investigated by source collision-induced dissociation (CID) in a heated capillary electrospray ion source. They were compared with the relative stabilities in solution obtained by thermal denaturation monitored by UV spectrophotometry. The results clearly show that both hydrogen bonding and base stacking interactions that are present in solution are maintained in the gas phase. This suggests that the electrospray process preserves the double-helix structure of DNA. A step by step opening of the double helix structure is proposed for the gas-phase dissociation, competing with the covalent bond cleavage of bases. We also draw attention to the fact that by source CID, it is the kinetic stability of the complexes that is probed. In particular, this implies that only complexes of the same size can be compared.

Gas phase thermal denaturation of an oligonucleotide duplex and its complexes with minor groove binders.

Electrospray ionization with in-source collisionally induced dissociation has been used to probe the gas phase stability of an oligonucleotide duplex and its complexes with some minor groove binding drugs. On the basis of the arguments developed in detail by Drahos et al. (J. Mass Spectrom. 1999; 34:1373), this type of experiment can also be described as 'thermal denaturation in the gas phase'. We found that the gas phase denaturation curves were very similar to the solution phase denaturation curves determined by the traditional UV spectrophotometric method and, by analogy with the melting temperature T(m) which characterizes the stability in solution, we define a melting voltage V(m) to characterize the stability in the gas phase. A comparison of the T(m) and V(m) relative values suggests that the structure of the complexes is conserved during the electrospray process which transfers the ions from the solution to the gas phase.

Interaction between antitumor drugs and a double-stranded oligonucleotide studied by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry was used to investigate the complex formation between a double-stranded oligonucleotide and various antitumor drugs belonging to two categories: intercalators (ethidium bromide, amsacrine and ascididemin) and minor groove binders (Hoechst 33258, netropsin, distamycin A, berenil and DAPI). The goal of this study was to determine whether the relative intensities in the mass spectra reflect the relative abundances of the species in the solution phase. The full-scan mass spectra suggest non-specific binding for the intercalators and specific binding for the minor groove binders. The preferential stoichiometries adopted by each minor groove binder were determined by studying the influence of the drug concentration on the spectra. We obtained 2:1 > 1:1 for distamycin, 1:1 > 2:1 for Hoechst 33258 and DAPI and only the 1 : 1 complex for netropsin and berenil. These features reflect their known behavior in solution. The compared tandem mass spectra of the 1 : 1 complexes with Hoechst 33258 and netropsin, when correlated with published crystallographic data, suggest the possibility of inferring some structural information. The relative binding affinities of the drug for the considered duplex were deduced with two by two competition experiments, assuming that the relative intensities reflect the composition of the solution phase. The obtained affinity scale is netropsin > distamycin A > DAPI > Hoechst 33258 > berenil. These examples show some of the potential uses of mass spectrometry as a useful tool for the characterization of specific drug binding to DNA, and possibly a rapid drug screening method requiring small amounts of materials.

Percutaneous autologous bone marrow grafting on the site of tibial delayed union.

Six months after injury, 150 mL of autogenous bone marrow was applied percutaneously at the site of delayed union to stimulate the healing of a tibial delayed union fracture in a 44 year-old man. Five months following the procedure, the fracture gaps and bone defects were completely filled with callus, the external fixator was removed, and the patient started using normal leg loading.

Paracetamol-propyphenazone interaction and formulation difficulties associated with eutectic formation in combination solid dosage forms.

Polymorphic behaviours of paracetamol and propyphenazone and interaction between these two compounds were investigated using differential scanning calorimetry (DSC), X-ray powder diffraction and Fourier transform-infrared (FT-IR)-spectroscopy. Binary mixtures containing various ratios of the compounds were prepared as physical and fused mixtures and analysed by DSC to study their thermal behaviours. Phase diagrams obtained from the melting endotherms of the binary mixtures demonstrated formation of an eutectic mixture at a paracetamol-propyphenazone combination of about 35:65 (w/w) with an eutectic temperature of 56 degrees C. The FT-IR spectroscopy revealed no chemical interaction due to eutectic formation, and a lower degree of crystallinity of the eutectic mixture than individual substances was observed by X-ray powder diffraction analysis. The DSC and X-ray powder diffraction data demonstrated a polymorphic change in propyphenazone as a result of melting of the compound. Tablets, containing both paracetamol and propyphenazone in a combination formulation and prepared using standard wet granulation technology, were found to have physical instability when packed in either polyvinylchloride// aluminium or polyvinylchloride/polyvinyldienechloride// aluminium blisters and stored for one month at 40 degrees C with either 75% relative humidity or without any humidity control. The instability of the tablets was more apparent under the high humidity condition.