Stacking Interactions and Flexibility of Human Telomeric Multimers.

G-quadruplexes (G4s) are helical four-stranded structures forming from guanine-rich nucleic acid sequences, which are thought to play a role in cancer development and malignant transformation. Most current studies focus on G4 monomers, yet under suitable and biologically relevant conditions, G4s undergo multimerization. Here, we investigate the stacking interactions and structural features of telomeric G4 multimers by means of a novel low-resolution structural approach that combines small-angle X-ray scattering (SAXS) with extremely coarse-grained (ECG) simulations. The degree of multimerization and the strength of the stacking interaction are quantitatively determined in G4 self-assembled multimers. We show that self-assembly induces a significant polydispersity of the G4 multimers with an exponential distribution of contour lengths, consistent with a step-growth polymerization. On increasing DNA concentration, the strength of the stacking interaction between G4 monomers increases, as well as the average number of units in the aggregates. We utilized the same approach to explore the conformational flexibility of a model single-stranded long telomeric sequence. Our findings indicate that its G4 units frequently adopt a beads-on-a-string configuration. We also observe that the interaction between G4 units can be significantly affected by complexation with benchmark ligands. The proposed methodology, which identifies the determinants that govern the formation and structural flexibility of G4 multimers, may be an affordable tool aiding in the selection and design of drugs that target G4s under physiological conditions.

The hope and hype of ellagic acid and urolithins as ligands of SARS-CoV-2 Nsp5 and inhibitors of viral replication.

Non-structural protein 5 (Nsp5) is a cysteine protease that plays a key role in SARS-CoV-2 replication, suppressing host protein synthesis and promoting immune evasion. The investigation of natural products as a potential strategy for Nsp5 inhibition is gaining attention as a means of developing antiviral agents. In this work, we have investigated the physicochemical properties and structure-activity relationships of ellagic acid and its gut metabolites, urolithins A-D, as ligands of Nsp5. Results allow us to identify urolithin D as promising ligand of Nsp5, with a dissociation constant in the nanomolar range of potency. Although urolithin D is able to bind to the catalytic cleft of Nsp5, the appraisal of its viral replication inhibition against SARS-CoV-2 in Vero E6 assay highlights a lack of activity. While these results are discussed in the framework of the available literature reporting conflicting data on polyphenol antiviral activity, they provide new clues for natural products as potential viral protease inhibitors.

Stability of Human Telomeric G-Quadruplexes Complexed with Photosensitive Ligands and Irradiated with Visible Light.

Guanine-rich DNA sequences can fold into non-canonical nucleic acid structures called G-quadruplexes (G4s). These nanostructures have strong implications in many fields, from medical science to bottom-up nanotechnologies. As a result, ligands interacting with G4s have attracted great attention as candidates in medical therapies, molecular probe applications, and biosensing. In recent years, the use of G4-ligand complexes as photopharmacological targets has shown significant promise for developing novel therapeutic strategies and nanodevices. Here, we studied the possibility of manipulating the secondary structure of a human telomeric G4 sequence through the interaction with two photosensitive ligands, DTE and TMPyP4, whose response to visible light is different. The effect of these two ligands on G4 thermal unfolding was also considered, revealing the occurrence of peculiar multi-step melting pathways and the different attitudes of the two molecules on the quadruplex stabilization.

Polymorphism and Ligand Binding Modulate Fast Dynamics of Human Telomeric G-Quadruplexes.

Telomeric G-quadruplexes (G4s) are promising targets in the design and development of anticancer drugs. Their actual topology depends on several factors, resulting in structural polymorphism. In this study, we investigate how the fast dynamics of the telomeric sequence AG3(TTAG3)3 (Tel22) depends on the conformation. By using Fourier transform Infrared spectroscopy, we show that, in the hydrated powder state, Tel22 adopts parallel and mixed antiparallel/parallel topologies in the presence of K+ and Na+ ions, respectively. These conformational differences are reflected in the reduced mobility of Tel22 in Na+ environment in the sub-nanosecond timescale, as probed by elastic incoherent neutron scattering. These findings are consistent with the G4 antiparallel conformation being more stable than the parallel one, possibly due to the presence of ordered hydration water networks. In addition, we study the effect of Tel22 complexation with BRACO19 ligand. Despite the quite similar conformation in the complexed and uncomplexed state, the fast dynamics of Tel22-BRACO19 is enhanced compared to that of Tel22 alone, independently of the ions. We ascribe this effect to the preferential binding of water molecules to Tel22 against the ligand. The present results suggest that the effect of polymorphism and complexation on the G4 fast dynamics is mediated by hydration water.

Stabilization of the Dimeric State of SARS-CoV-2 Main Protease by GC376 and Nirmatrelvir.

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1' and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.

Role of fast dynamics in the complexation of G-quadruplexes with small molecules.

G-quadruplexes (G4s) formed by the human telomeric sequence AG3 (TTAG3)3 (Tel22) play a key role in cancer and aging. We combined elastic incoherent neutron scattering (EINS) and quasielastic incoherent neutron scattering (QENS) to characterize the internal dynamics of Tel22 G4s and to assess how it is affected by complexation with two standard ligands, Berberine and BRACO19. We show that the interaction with the two ligands induces an increase of the overall mobility of Tel22 as quantified by the mean squared displacements (MSD) of hydrogen atoms. At the same time, the complexes display a lower stiffness than G4 alone. Two different types of motion characterize the G4 nanosecond timescale dynamics. Upon complexation, an increasing fraction of G4 atomic groups participate in this fast dynamics, along with an increase in the relevant characteristic length scales. We suggest that the entropic contribution to the conformational free energy of these motions might be crucial for the complexation mechanisms.

Solvent Vibrations as a Proxy of the Telomere G-Quadruplex Rearrangements across Thermal Unfolding.

G-quadruplexes (G4s) are noncanonical forms of DNA involved in many key genome functions. Here, we exploited UV Resonance Raman scattering to simultaneously explore the vibrational behavior of a human telomeric G4 (Tel22) and its aqueous solvent as the biomolecule underwent thermal melting. We found that the OH stretching band, related to the local hydrogen-bonded network of a water molecule, was in strict relation with the vibrational features of the G4 structure as a function of temperature. In particular, the modifications to the tetrahedral ordering of the water network were strongly coupled to the DNA rearrangements, showing changes in temperature that mirrored the multi-step melting process of Tel22. The comparison between circular dichroism and Raman results supported this view. The present findings provide novel insights into the impact of the molecular environment on G4 conformation. Improving current knowledge on the solvent structural properties will also contribute to a better understanding of the role played by water arrangement in the complexation of G4s with ligands.

Amyloid Self-Assembly of Lysozyme in Self-Crowded Conditions: The Formation of a Protein Oligomer Hydrogel.

A method is designed to quickly form protein hydrogels, based on the self-assembly of highly concentrated lysozyme solutions in acidic conditions. Their properties can be easily modulated by selecting the curing temperature. Molecular insights on the gelation pathway, derived by in situ FTIR spectroscopy, are related to calorimetric and rheological results, providing a consistent picture on structure-property correlations. In these self-crowded samples, the thermal unfolding induces the rapid formation of amyloid aggregates, leading to temperature-dependent quasi-stationary levels of antiparallel cross ß-sheet links, attributed to kinetically trapped oligomers. Upon subsequent cooling, thermoreversible hydrogels develop by the formation of interoligomer contacts. Through heating/cooling cycles, the starting solutions can be largely recovered back, due to oligomer-to-monomer dissociation and refolding. Overall, transparent protein hydrogels can be easily formed in self-crowding conditions and their properties explained, considering the formation of interconnected amyloid oligomers. This type of biomaterial might be relevant in different fields, along with analogous systems of a fibrillar nature more commonly considered.

Structure of human telomere G-quadruplex in the presence of a model drug along the thermal unfolding pathway.

A multi-technique approach, combining circular dichroism spectroscopy, ultraviolet resonance Raman spectroscopy and small angle scattering techniques, has been deployed to elucidate how the structural features of the human telomeric G-quadruplex d[A(GGGTTA)3GGG] (Tel22) change upon thermal unfolding. The system is studied both in the free form and when it is bound to Actinomycin D (ActD), an anticancer ligand with remarkable conformational flexibility. We find that at room temperature binding of Tel22 with ActD involves end-stacking upon the terminal G-tetrad. Structural evidence for drug-driven dimerization of a significant fraction of the G-quadruplexes is provided. When the temperature is raised, both free and bound Tel22 undergo melting through a multi-state process. We show that in the intermediate states of Tel22 the conformational equilibrium is shifted toward the (3+1) hybrid-type, while a parallel structure is promoted in the complex. The unfolded state of the free Tel22 is consistent with a self-avoiding random-coil conformation, whereas the high-temperature state of the complex is observed to assume a quite compact form. Such an unprecedented high-temperature arrangement is caused by the persistent interaction between Tel22 and ActD, which stabilizes compact conformations even in the presence of large thermal structural fluctuations.

Trehalose-induced slowdown of lysozyme hydration dynamics probed by EDLS spectroscopy.

We use extended depolarized light scattering spectroscopy to study the dynamics of water in a lysozyme-trehalose aqueous solution over a broad time scale, from hundreds to fractions of picoseconds. We provide experimental evidence that the sugar, present in the ternary solution in quantity relevant for biopreservation, strongly modifies the solvation properties of the protein. By comparing aqueous solutions of lysozyme with and without trehalose, we show that the combined action of sugar and protein produces an exceptional dynamic slowdown of a fraction of water molecules around the protein, which become more than twice slower than in the absence of trehalose. We speculate that this ultraslow water may be caged between the sugar and protein surface, consistently with a water entrapment scenario. We also demonstrate that the dynamics of these water molecules gets slower and slower upon cooling. On the basis of these findings, we believe such ultraslow water close to the lysozyme is likely to be involved in the mechanism of bioprotection.

On the interplay between the slowdown of dynamics and the kinetics of aggregation: The case study of a reactive binary mixture.

Modeling the kinetics of aggregation requires a proper strategy to take into account not only the reactivity of reagents but also the ability they have to diffuse. The lack of direct information about diffusion represents the most serious experimental obstacle to the use of diffusion-corrected mean-field equations, which is usually overcome by using information on the structural relaxation dynamics. A very accurate description of the entire kinetics of aggregation can be made by introducing a single time scale of diffusion, set by the structural relaxation time τ of the system according to ∼τ(ξ), with ξ a fractional exponent. Here, we apply this modeling to the case of a reactive binary mixture made of diglycidyl ether of bisphenol-A and 1,3-phenylenediamine, where the reaction proceeds along an autocatalyic (hydroxyl catalyzed) and a non-catalytic (impurity catalyzed) pathway and find that a very small value of the exponent ξ = 0.27 ± 0.03 is needed to reproduce all the data. Our results help revise some preconceived ideas: contrary to widely held assumptions, we find that (i) the time scale of diffusion neither increases proportionally to the structural relaxation time nor is related to τ by a power law with the same fractional exponent as that relating τ to conductivity; (ii) no direct connection exists between the transition to diffusion-control and the development of a gel network or formation of a glassy phase; and (iii) there is no significant difference in the enthalpy barrier for bond formation in the presence of hydroxyl or other than hydroxyl catalyst groups.

Concentration dependence of hydration water in a model peptide.

The molecular dynamics of aqueous solutions of a model amphiphilic peptide is studied as a function of concentration by broad-band light scattering experiments. Similarly to protein aqueous solutions, a considerable retardation, of about a factor 6-8, of hydration water dynamics with respect to bulk water is found, showing a slight dependence on solute concentration. Conversely, the average number of water molecules perturbed by the presence of peptide, i.e. the hydration number, appears to be strongly modified by adding solute. Its behaviour, decreasing upon increasing concentration, can be interpreted considering the random close-to-contact condition experienced by solute particles. Overall, the present findings support the view of a "long range" effect of peptides on the surrounding water, extending beyond the first two hydration shells.

Ultraviolet Resonant Raman Scattering of Electrolyte Solutions.

Saltwater stands as the most prevalent liquid on Earth. Consequently, substantial interest has been directed toward its characterization, both as an independent system and as a solvent for complex structures such as biomacromolecules. In the last few decades, special emphasis was placed on the investigation of the hydration properties of ions for the fundamental role they play in numerous chemical processes. In this study, we employed multi-wavelength Raman spectroscopy to examine the hydration shell surrounding bromide ions in solutions of simple electrolytes, specifically lithium bromide, potassium bromide, and cesium bromide, at two different concentrations. Cation-induced differences among electrolytes were observed in connection to their tendency to form ion pairs. An increased sensitivity to reveal the structure of the first hydration shell was evidenced when employing ultraviolet excitation in the 228-266 nm range, under resonance conditions with the charge transfer transition to the solvent peaked at about 200 nm. Other than a significant increase in the Raman cross-section for the OH stretching band when shifting from pure water to the solution, a larger enhancement for the Raman signal of the H-O-H bending mode over the stretching vibration was observed. Thus, the bending band plays a crucial role in monitoring the H-bond structure of water around the anions related to the charge distribution within the first hydration shell of anions, being an effective probe of hydration phenomena.

Molecular mechanisms behind BRACO19 and human telomeric G-quadruplex interaction.

Human telomeres (HTs) can form DNA G-quadruplex (G4), an attractive target for anticancer and antiviral drugs. HT-G4s exhibit inherent structural polymorphism, posing challenges for understanding their specific recognition by ligands. Here, we aim to explore the impact of different topologies within a small segment of the HT (Tel22) on its interaction with BRACO19, a rationally designed G4 ligand with high quadruplex affinity, already employed in in-vivo treatments. Our multi-technique approach is based on the combined use of a set of contactless spectroscopic tools. Circular dichroism and UV resonance Raman spectroscopy probe ligand-induced conformational changes in the G4 sequence, while UV-visible absorption, coupled with steady-state fluorescence spectroscopy, provides further insights into the electronic features of the complex, exploiting the photoresponsive properties of BRACO19. Overall, we find that modifying the topology of the unbound Tel22 through cations (K+ or Na+), serves as a critical determinant for ligand interactions and binding modes, thus influencing the HT-G4's assembly capabilities. Furthermore, we show how fluorescence serves as a valuable probe for recognizing cation-driven multimeric structures, which may be present in living organisms, giving rise to pathological forms.

Dynamic Personality of Proteins and Effect of the Molecular Environment.

Protein dynamics display distinct traits that are linked to their specific biological function. However, the interplay between intrinsic dynamics and the molecular environment on protein stability remains poorly understood. In this study, we investigate, by incoherent neutron scattering, the subnanosecond time scale dynamics of three model proteins: the mesophilic lysozyme, the thermophilic thermolysin, and the intrinsically disordered ß-casein. Moreover, we address the influence of water, glycerol, and glucose, which create progressively more viscous matrices around the protein surface. By comparing the protein thermal fluctuations, we find that the internal dynamics of thermolysin are less affected by the environment compared to lysozyme and ß-casein. We ascribe this behavior to the protein dynamic personality, i.e., to the stiffer dynamics of the thermophilic protein that contrasts the influence of the environment. Remarkably, lysozyme and thermolysin in all molecular environments reach a critical common flexibility when approaching the calorimetric melting temperature.

Correction to "Electrospun Nanofibrous UV Filters with Bidirectional Actuation Properties Based on Salmon Sperm DNA/Silk Fibroin for Biomedical Applications".

[This corrects the article DOI: 10.1021/acsomega.3c04563.].

Volume properties and spectroscopy: a terahertz Raman investigation of hen egg white lysozyme.

The low frequency depolarized Raman spectra of 100 mg/ml aqueous solutions of hen egg white lysozyme (HEWL) have been collected in the 25-85 °C range. Short and long exposures to high temperatures have been used to modulate the competition between the thermally induced reversible and irreversible denaturation processes. A peculiar temperature evolution of spectra is evidenced under prolonged exposure of the protein solution at temperatures higher than 65 °C. This result is connected to the self-assembling of polypeptide chains and testifies the sensitivity of the technique to the properties of both protein molecule and its surrounding. Solvent free spectra have been obtained after subtraction of elastic and solvent components and assigned to a genuine vibrational contribution of hydrated HEWL. A straight similarity is observed between the solvent-free THz Raman feature and the vibrational density of states as obtained by molecular dynamics simulations; according to this, we verify the relation between this spectroscopic observable and the effective protein volume, and distinguish the properties of this latter respect to those of the hydration shell in the pre-melting region.

Development of Salmon Sperm DNA/Regenerated Silk Bio-Based Films for Biomedical Studies on Human Keratinocyte HaCaT Cells under Solar Spectrum.

In this study, we fabricated adhesive patches from silkworm-regenerated silk and DNA to safeguard human skin from the sun's rays. The patches are realized by exploiting the dissolution of silk fibers (e.g., silk fibroin (SF)) and salmon sperm DNA in formic acid and CaCl2 solutions. Infrared spectroscopy is used to investigate the conformational transition of SF when combined with DNA; the results indicated that the addition of DNA provides an increase in the SF crystallinity. UV-Visible absorption and circular dichroism spectroscopy showed strong absorption in the UV region and the presence of B-form of DNA once dispersed in the SF matrix, respectively. Water absorption measurements as well as thermal dependence of water sorption and thermal analysis, suggested the stability of the fabricated patches. Biological results on cellular viability (MTT assay) of keratinocyte HaCaT cells after exposures to the solar spectrum showed that both SF and SF/DNA patches are photo-protective by increasing the cellular viability of keratinocytes after UV component exposure. Overall, these SF/DNA patches promise applications in wound dressing for practical biomedical purposes.

Electrospun Nanofibrous UV Filters with Bidirectional Actuation Properties Based on Salmon Sperm DNA/Silk Fibroin for Biomedical Applications.

In this study, we dissolved Bombyx mori degummed silk [i.e., silk fibroin (SF)] and salmon sperm deoxyribonucleic acid (DNA) in water and used a bioinspired spinning process to obtain an electrospun nanofibrous SF-based patch (ESF). We investigated the bidirectional macroscale actuation behavior of ESF in response to water vapor and its UV-blocking properties as well as those of ESF/DNA films. Fourier transform infrared (FTIR) results suggest that the formation of ß-sheet-rich structures promotes the actuation effect. ESF/DNA film with high-ordered and ß-sheet-rich structures exhibits higher electrical conductivity and is water-insoluble. Given the intrinsic ability of both SF and DNA to absorb UV radiation, we performed biological experiments on the viability of keratinocyte HaCaT cells after exposure to solar spectrum components. Our findings indicate that the ESF/DNA patch is photoprotective and can increase the cellular viability of keratinocytes after UV exposure. Furthermore, we demonstrated that ESF/DNA patches treated with water vapor can serve as suitable scaffolds for tissue engineering and can improve tissue regeneration when cellularized with HaCaT cells. The 3D shape morphing capability of these patches, along with their potential as UV filters, could offer significant practical advantages in tissue engineering.

Hydration Dynamics of Model Peptides with Different Hydrophobic Character.

The multi-scale dynamics of aqueous solutions of the hydrophilic peptide N-acetyl-glycine-methylamide (NAGMA) have been investigated through extended frequency-range depolarized light scattering (EDLS), which enables the broad-band detection of collective polarizability anisotropy fluctuations. The results have been compared to those obtained for N-acetyl-leucinemethylamide (NALMA), an amphiphilic peptide which shares with NAGMA the same polar backbone, but also contains an apolar group. Our study indicates that the two model peptides induce similar effects on the fast translational dynamics of surrounding water. Both systems slow down the mobility of solvating water molecules by a factor 6-8, with respect to the bulk. Moreover, the two peptides cause a comparable far-reaching spatial perturbation extending to more than two hydration layers in diluted conditions. The observed concentration dependence of the hydration number is explained considering the random superposition of different hydration shells, while no indication of solute aggregation phenomena has been found. The results indicate that the effect on the dynamics of water solvating the amphiphilic peptide is dominated by the hydrophilic backbone. The minor impact of the hydrophobic moiety on hydration features is consistent with structural findings derived by Fourier transform infrared (FTIR) measurements, performed in attenuated total reflectance (ATR) configuration. Additionally, we give evidence that, for both systems, the relaxation mode in the GHz frequency range probed by EDLS is related to solute rotational dynamics. The rotation of NALMA occurs at higher timescales, with respect to the rotation of NAGMA; both processes are significantly slower than the structural dynamics of hydration water, suggesting that solute and solvent motions are uncoupled. Finally, our results do not indicate the presence of super-slow water (relaxation times in the order of tens of picoseconds) around the peptides investigated.