Conformational plasticity of DNA secondary structures: probing the conversion between i-motif and hairpin species by circular dichroism and ultraviolet resonance Raman spectroscopies.

The promoter regions of important oncogenes such as BCL2 and KRAS contain GC-rich sequences that can form distinctive noncanonical DNA structures involved in the regulation of transcription: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. Interestingly, BCL2 and KRAS promoter i-motifs are highly dynamic in nature and exist in a pH-dependent equilibrium with hairpin and even with hybrid i-motif/hairpin species. Herein, the effects of pH and presence of cell-mimicking molecular crowding conditions on conformational equilibria of the BCL2 and KRAS i-motif-forming sequences were investigated by ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopies. Multivariate analysis of CD data was essential to model the presence and identity of the species involved. Analysis of UVRR spectra measured as a function of pH, performed also by the two-dimensional correlation spectroscopy (2D-COS) technique, showed the role of several functional groups in the DNA conformational transitions, and provided structural and dynamic information. Thus, the UVRR investigation of intramolecular interactions and of local and environmental dynamics in promoting the different species induced by the solution conditions provided valuable insights into i-motif conformational transitions. The combined use of the two spectroscopic tools is emphasized by the relevant possibility of working in the same DNA concentration range and by the heterospectral UVRR/CD 2D-COS analysis. The results of this study shed light on the factors that can influence at the molecular level the equilibrium between the different conformational species putatively involved in the oncogene expression.

Ligand binding to G-quadruplex DNA: new insights from ultraviolet resonance Raman spectroscopy.

G-Quadruplexes (G4s) are noncanonical nucleic acid structures involved in the regulation of several biological processes of many organisms. The rational design of G4-targeting molecules developed as potential anticancer and antiviral therapeutics is a complex problem intrinsically due to the structural polymorphism of these peculiar DNA structures. The aim of the present work is to show how Ultraviolet Resonance Raman (UVRR) spectroscopy can complement other techniques in providing valuable information about ligand/G4 interactions in solution. Here, the binding of BRACO-19 and Pyridostatin - two of the most potent ligands - to selected biologically relevant G4s was investigated by polarized UVRR scattering at 266 nm. The results give new insights into the binding mode of these ligands to G4s having different sequences and topologies by performing an accurate analysis of peaks assigned to specific groups and their changes upon binding. Indeed, the UVRR data not only show that BRACO-19 and Pyridostatin interact with different G4 sites, but also shed light on the ligand and G4 chemical groups really involved in the interaction. In addition, UVRR results complemented by circular dichroism data clearly indicate that the binding mode of a ligand can also depend on the conformation(s) of the target G4. Overall, these findings demonstrate the utility of using UVRR spectroscopy in the investigation of G4s and G4-ligand interactions in solution.

Crowding and conformation interplay on human DNA G-quadruplex by ultraviolet resonant Raman scattering.

The G-quadruplex-forming telomeric sequence (TTAGGG)4TT was investigated by polarized Ultraviolet Resonance Raman Scattering (UVRR) at 266 nm. The presence of 40% poly(ethylene glycol) and the so-called "self-crowding" condition were used to induce the hybrid-to-parallel topology transition. Analysis of frequency shifts with temperature showed the role of several functional groups in the topological transitions and provides structural dynamical information. Circular dichroism under similar conditions was used as a reference. UVRR shed light on the effect of intramolecular interactions and of local and environmental dynamics in promoting different G-quadruplex topologies, induced by solution conditions or by temperature changes. Overall, these findings showed the enormous potential of this spectroscopy for G-quadruplex conformational studies.

Myelography Iodinated Contrast Media. 2. Conformational Versatility of Iopamidol in the Solid State.

The phenomenon of polymorphism is of great relevance in pharmaceutics, since different polymorphs have different physicochemical properties, e.g., solubility, hence, bioavailability. Coupling diffractometric and spectroscopic experiments with thermodynamic analysis and computational work opens to a methodological approach which provides information on both structure and dynamics in the solid as well as in solution. The present work reports on the conformational changes in crystalline iopamidol, which is characterized by atropisomerism, a phenomenon that influences both the solution properties and the distinct crystal phases. The conformation of iopamidol is discussed for three different crystal phases. In the anhydrous and monohydrate crystal forms, iopamidol molecules display a syn conformation of the long branches stemming out from the triiodobenzene ring, while in the pentahydrate phase the anti conformation is found. IR and Raman spectroscopic studies carried out on the three crystal forms, jointly with quantum chemical computations, revealed that the markedly different spectral features can be specifically attributed to the different molecular conformations. Our results on the conformational versatility of iopamidol in different crystalline phases, linking structural and spectroscopic evidence for the solution state and the solid forms, provide a definite protocol for grasping the signals that can be taken as conformational markers. This is the first step for understanding the crystallization mechanism occurring in supersaturated solution of iopamidol molecules.

Cytosine epigenetic modifications and conformational changes in G-quadruplex DNA: An ultraviolet resonance Raman spectroscopy study.

Epigenetic modifications of DNA are known to play important regulatory roles in biological systems, especially in regulation of gene expression, and are associated with many types of human diseases, including cancer. Alternative DNA secondary structures, such as G-quadruplexes, can also influence gene transcription, thus suggesting that such structures may represent a distinctive layer of epigenetic information. G-quadruplex structures and DNA epigenetic modifications often go side by side, and recent evidence reveals that cytosine modifications within loops of G-quadruplexes can play a role in modulating their stability and structural polymorphism. Therefore, the development and validation of experimental techniques that can easily and reliably analyse G-quadruplex structures are highly desirable. In the present study, we propose to exploit the advantages of UV resonance Raman (UVRR) spectroscopy to investigate cytosine epigenetic modifications along with conformational changes in G-quadruplex-forming DNA. Our findings show that clear and specific spectral changes occur when there is a change in a G-quadruplex structure. Moreover, UVRR spectral analysis can indirectly distinguish the spectral variations occurring because of modifications in the guanine glycosidic conformations, as well as detect changes in the loops induced by H-bond formation or hydration of nitrogenous bases. The results further underscore the utility of UVRR spectroscopy for G-quadruplex structure elucidation under biologically relevant solution conditions.

Ligand-based drug repurposing strategy identified SARS-CoV-2 RNA G-quadruplex binders.

The single-stranded RNA genome of SARS-CoV-2 contains some G-quadruplex-forming G-rich elements which are putative drug targets. Here, we performed a ligand-based pharmacophore virtual screening of FDA approved drugs to find candidates targeting such RNA structures. Further in silico and in vitro assays identified three drugs as emerging SARS-CoV-2 RNA G-quadruplex binders.

Prediction of the transmission through thin-film waveguides for X-ray microscopy.

Thin-film slab waveguides can confine incident X-ray beams in one direction in guiding layers as thin as 10 nm. Consequently they can provide attractive beam dimensions for microscopy purposes. This report presents a simple model and analytical equations for the transmission calculation, which provide results consistent with the rigorous calculations based on recursion techniques. By using these results the waveguide transmission can be compared directly with other microscopy objectives. Ideally X-ray waveguides can filter the spatially coherent content out of an incident radiation beam with an efficiency of 1. The transmissions measured for state-of-the-art one- and two-dimensional waveguides are found to correspond to experimental efficiencies of 0.5 for each confinement direction. Waveguides with thinner guiding layers cannot be used efficiently in highly collimated beams; instead the beam divergence in unfocused beamlines at state-of-the-art synchrotron radiation sources may eventually have to be increased to the larger angular acceptance of these waveguides by use of other focusing optics.