Investigation of genomic DNA methylation by ultraviolet resonant Raman spectroscopy

Cytosine plays a preeminent role in DNA methylation, an epigenetic mechanism that regulates gene expression, the misregulation of which can lead to severe diseases. Several methods are nowadays employed for assessing the global DNA methylation levels, but none of them combines simplicity, high sensitivity, and low operating costs to be translated into clinical applications. Ultraviolet (UV) resonant Raman measurements at excitation wavelengths of 272 nm, 260 nm, 250 nm, and 228 nm have been carried out on isolated deoxynucleoside triphosphates (dNTPs), on a dNTP mixture as well as on genomic DNA (gDNA) samples, commercial from salmon sperm and non-commercial from B16 murine melanoma cell line. The 228 nm excitation wavelength was identified as the most suitable energy for enhancing cytosine signals over the other DNA bases. The UV Raman measurements performed at this excitation wavelength on hyper-methylated and hypo-methylated DNA from Jurkat leukemic T-cell line have revealed significant spectral differences with respect to gDNA isolated from salmon sperm and mouse melanoma B16 cells. This demonstrates how the proper choice of the excitation wavelength, combined with optimized extraction protocols, makes UV Raman spectroscopy a suitable technique for highlighting the chemical modifications undergone by cytosine nucleotides in gDNA upon hyper- and hypo-methylation events

The mixed longitudinal–transverse nature of collective modes in water

We report high-resolution, high-statistics inelastic x-ray scattering measurements of the dynamic structure factor of water as a function of momentum and energy transfer in various thermodynamic conditions, including high-pressure liquid near the melting point, supercooled liquid and polycrystalline ice. For momentum transfer values below 8 nm−1, two collective excitations associated with longitudinal and transverse acoustic modes were observed. Above 8 nm−1, another excitation was detected in the liquid. Comparison with polycrystalline data and molecular dynamics simulations suggests that this mode is related to longitudinal–transverse mixing of mode symmetry

Oxidative damage in DNA bases revealed by UV resonant Raman spectroscopy

We report on the use of the UV Raman technique to monitor the oxidative damage of deoxynucleotide triphosphates (dATP, dGTP, dCTP and dTTP) and DNA (plasmid vector) solutions. Nucleotide and DNA aqueous solutions were exposed to hydrogen peroxide (H2O2) and iron containing carbon nanotubes (CNTs) to produce Fenton's reaction and induce oxidative damage. UV Raman spectroscopy is shown to be maximally efficient to reveal changes in the nitrogenous bases during the oxidative mechanisms occurring on these molecules. The analysis of Raman spectra, supported by numerical computations, revealed that the Fenton's reaction causes an oxidation of the nitrogenous bases in dATP, dGTP and dCTP solutions leading to the production of 2-hydroxyadenine, 8-hydroxyguanine and 5-hydroxycytosine. No thymine change was revealed in the dTTP solution under the same conditions. Compared to single nucleotide solutions, plasmid DNA oxidation has resulted in more radical damage that causes the breaking of the adenine and guanine aromatic rings. Our study demonstrates the advantage of using UV Raman spectroscopy for rapidly monitoring the oxidation changes in DNA aqueous solutions that can be assigned to specific nitrogenous bases

Combining Raman and infrared spectroscopy as a powerful tool for the structural elucidation of cyclodextrin-based polymeric hydrogels

A detailed experimental and theoretical vibrational analysis of hydrogels of b-cyclodextrin nanosponges (b-CDNS), obtained by polymerization of b-cyclodextrin (b-CD) with the cross-linking agent ethylenediaminetetraacetic acid (EDTA), is reported here. Thorough structural characterization is achieved by exploiting the complementary selection rules of FTIR-ATR and Raman spectroscopies and by supporting the spectral assignments by DFT calculations of the spectral profiles. The combined analysis of the FTIR-ATR spectra of the polymers hydrated with H2O and D2O allowed us to isolate the HOH bending of water molecules not involved in symmetrical, tetrahedral environments. The analysis of the HOH bending mode was carried out as a function of temperature, showing the existence of a supercooled state of the water molecules. The highest level of cooperativity of the hydrogen bond scheme was reached at a value of the b-CD/EDTA molar ratio n = 6. Finally, the connectivity pattern of ‘‘uncoupled’’ water molecules bound to the nanosponge backbone was found to be weakened by increasing T. The temperature above which the population of non-tetracoordinated water molecules becomes predominant turned out to be independent of the parameter n

Short-wavelength four wave mixing experiments using single and two-color schemes at FERMI

The development of ultra-bright extreme ultraviolet (EUV) and X-ray free electron laser (FEL) sources has enabled the extension of wave-mixing approaches into the short wavelength regime. Such a class of experiments relies upon nonlinear interactions among multiple light pulses offering a unique tool for exploring the dynamics of ultrafast processes and correlations between selected excitations at relevant length and time scales adding elemental and site selectivity as well. Besides the availability of a suitable photon source, the implementation of wave mixing methodology requires efforts in developing the instrumental set-up. We have realized at the FERMI FEL two dedicated set-ups to handle multiple FEL beams with preselected parameters in a non-collinear fashion and control their interaction sequence at the target. These unique apparatuses, combined with the exceptional characteristics of the seeded FERMI FEL, have allowed us to make the first steps into this field and further advances are foreseen in the near future

Free electron laser-driven ultrafast rearrangement of the electronic structure in Ti

High-energy density extreme ultraviolet radiation delivered by the FERMI seeded free-electron laser has been used to create an exotic nonequilibrium state of matter in a titanium sample characterized by a highly excited electron subsystem at temperatures in excess of 10 eV and a cold solid-density ion lattice. The obtained transient state has been investigated through ultrafast absorption spectroscopy across the Ti M2,3-edge revealing a drastic rearrangement of the sample electronic structure around the Fermi level occurring on a time scale of about 100 fs

A novel free-electron laser single-pulse Wollaston polarimeter for magneto-dynamical studies

Here, we report on the conceptual design, the hardware realization, and the first experimental results of a novel and compact x-ray polarimeter capable of a single-pulse linear polarization angle detection in the extreme ultraviolet photon energy range. The polarimeter is tested by performing time resolved pump-probe experiments on a Ni80Fe20 Permalloy film at the M-2,M-3 Ni edge at an externally seeded free-electron laser source. Comparison with similar experiments reported in the literature shows the advantages of our approach also in view of future experiments

Probing the molecular connectivity of water confined in polymer hydrogels

The molecular connectivity and the extent of hydrogen-bond patterns of water molecules confined in the polymer hydrogels, namely cyclodextrin nanosponge hydrogels, are here investigated by using vibrational spectroscopy experiments. The proposed spectroscopic method exploits the combined analysis of the vibrational spectra of polymers hydrated with water and deuterated water that allows us to separate and selectively investigate the temperature-evolution of the HOH bending mode of engaged water molecules and of the vibrational modes assigned to specific chemical groups of the polymer matrix involved in the physical interactions with water. As main results, we find a strong experimental evidence of a liquid-like behaviour of water molecules confined in the nano-cavities of hydrogel and we observe a characteristic destructuring effect on the hydrogen-bonds network of confined water induced by thermal motion. More interestingly, the extent of this temperature-disruptive effect is found to be selectively triggered by the cross-linking degree of the hydrogel matrix. These results give a more clear picture of the molecular mechanism of water confinement in the pores of nanosponge hydrogel and open the possibility to exploit the spectroscopic method here proposed as investigating tools for water-retaining soft materials