Here we report the synthesis of two new 13C thymidine (T) isotopes labeled at two key positions in its structure, C2═O and C4═O, and the first IR spectra of each T isotope in H2O and in D2O. Our experimental isotope shifts and normal mode calculations indicate some structurally sensitive modes may be more localized than previously thought, making them regiosensitive probes of thymidine's chemical environment. These results provide new physical insight into the normal modes of thymidine, and will guide future studies to determine how these structurally and environmentally sensitive probe vibrations are impacted by the duplex environment.

The structures and energetics of nucleic acid assemblies and enzyme-substrate complexes are of intense interest. New methods to probe the contributions of individual components under a given set of conditions are needed. Vibrational states that include known groups of atoms contained in a structure can be sensitive probes of local structure in nucleic or amino acid sequences, and of their fractional contributions to total stability. In this work we calibrate the thymine C2=O stretch via developing nucleobase isotope edited variable temperature infrared-based methods as a "molecular thermometer" to measure melting transitions at a single base pair in a duplexed DNA sequence.

S-adenosyl-L-methionine (SAM) is an abundant biomolecule used by methyltransferases to regulate a wide range of essential cellular processes such as gene expression, cell signaling, protein functions, and metabolism. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off -target effects. Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between several states when free in solution. Infrared spectroscopy can detect different conformers of molecules if present in appreciable populations. When SAM is noncovalently bound within enzyme active sites, the nature and the number of different conformations of the molecule are likely to be altered from when it is free in solution. If there are unique structures or different numbers of conformers between different methyltransferase active sites, solution -state information may provide promising structural leads to increase inhibitor specificity for a particular methyltransferase. Toward this goal, frequencies measured in SAM's infrared spectra must be assigned to the motions of specific atoms via isotope incorporation at discrete positions. The incorporation of isotopes into SAM's structure can be accomplished via an established enzymatic synthesis using isotopically labeled precursors. However, published protocols produced an intense and highly variable IR signal which overlapped with many of the signals from SAM rendering comparison between isotopes challenging. We observed this intense absorption to be from co -purifying salts and the SAM counterion, producing a strong, broad signal at 1100 cm-1. Here, we report a revised SAM purification protocol that mitigates the contaminating salts and present the first IR spectra of isotopically labeled CD3-SAM. These results provide a foundation for isotopic labeling experiments of SAM that will define which atoms participate in individual molecular vibrations, as a means to detect specific molecular conformations.

Experimental methods to determine transition temperatures for individual base pair melting events in DNA duplexes are lacking despite intense interest in these thermodynamic parameters. Here, we determine the dimensions of the thymine (T) C2=O stretching vibration when it is within the DNA duplex via iso-topic substitutions at other atomic positions in the structure. After determining that this IR difference probe was localized enough to measure sub-molecular scale structures in high molecular weight complexes, we used this probe to develop a new isotope edited variable temperature infrared method to measure melting at various locations in a DNA structure. As an initial test of this “sub-molecular-scale thermometer”, we first applied our T13C2 difference infrared signal to measure location-dependent melting temperatures (TmL) in a DNA duplex via variable temperature attenuated total reflectance Fourier transform infrared (VT-ATR-FTIR) spectroscopy. We report that the TmL of a single Watson-Crick A-T base pair at the end of an A-T rich duplex is ~34.9±0.7˚C. This is slightly lower than the TmL of a single base pair at the middle position of the duplex (TmL ~35.6±0.2˚C), providing direct physical evidence for end fraying in A-T-rich DNA sequences.

DNA helicase activity is essential for the vital DNA metabolic processes of recombination, replication, transcription, translation, and repair. Recently, an unexpected, rapid exponential ATP-stimulated DNA unwinding rate was observed from an Archaeoglobus fulgidus helicase (AfXPB) as compared to the slower conventional helicases from Sulfolobus tokodaii, StXPB1 and StXPB2. This unusual rapid activity suggests a "molecular wrench" mechanism arising from the torque applied by AfXPB on the duplex structure in transitioning from open to closed conformations. However, much remains to be understood. Here, we investigate the concentration dependence of DNA helicase binding and ATP-stimulated kinetics of StXPB2 and AfXPB, as well as their binding and activity in Bax1 complexes, via an electrochemical assay with redox-active DNA monolayers. StXPB2 ATP-stimulated activity is concentration-independent from 8 to 200 nM. Unexpectedly, AfXPB activity is concentration-dependent in this range, with exponential rate constants varying from seconds at concentrations greater than 20 nM to thousands of seconds at lower concentrations. At 20 nM, rapid exponential signal decay ensues, linearly reverses, and resumes with a slower exponential decay. This change in AfXPB activity as a function of its concentration is rationalized as the crossover between the fast molecular wrench and slower conventional helicase modes. AfXPB-Bax1 inhibits rapid activity, whereas the StXPB2-Bax1 complex induces rapid kinetics at higher concentrations. This activity is rationalized with the crystal structures of these complexes. These findings illuminate the different physical models governing molecular wrench activity for improved biological insight into a key factor in DNA repair.

Structural bioinspiration in modern material science and biomimetics represents an actual trend that was originally based on the bioarchitectural diversity of invertebrate skeletons, specifically, honeycomb constructs of natural origin, which have been in humanities focus since ancient times. We conducted a study on the principles of bioarchitecture regarding the unique biosilica-based honeycomb-like skeleton of the deep-sea glass sponge Aphrocallistes beatrix. Experimental data show, with compelling evidence, the location of actin filaments within honeycomb-formed hierarchical siliceous walls. Principles of the unique hierarchical organization of such formations are discussed. Inspired by poriferan honeycomb biosilica, we designed diverse models, including 3D printing, using PLA-, resin-, and synthetic-glass-prepared corresponding microtomography-based 3D reconstruction.

The carbonyl stretching modes have been widely used in linear and two-dimensional infrared (IR) spectroscopy to probe the conformation, interaction, and biological functions of nucleic acids. However, due to their universal appearance in nucleobases, the IR absorption bands of nucleic acids are often highly congested in the 1600-1800 cm-1 region. Following the fruitful applications in proteins, 13C isotope labels have been introduced to the IR measurements of oligonucleotides to reveal their site-specific structural fluctuations and hydrogen bonding conditions. In this work, we combine recently developed frequency and coupling maps to develop a theoretical strategy that models the IR spectra of oligonucleotides with 13C labels directly from molecular dynamics simulations. We apply the theoretical method to nucleoside 5'-monophosphates and DNA double helices and demonstrate how elements of the vibrational Hamiltonian determine the spectral features and their changes upon isotope labeling. Using the double helices as examples, we show that the calculated IR spectra are in good agreement with experiments and the 13C isotope labeling technique can potentially be applied to characterize the stacking configurations and secondary structures of nucleic acids.

The level of interest in probing the strength of noncovalent interactions in DNA duplexes is high, as these weak forces dictate the range of suprastructures the double helix adopts under different conditions, in turn directly impacting the biological functions and industrial applications of duplexes that require making and breaking them to access the genetic code. However, few experimental tools can measure these weak forces embedded within large biological suprastructures in the native solution environment. Here, we develop experimental methods for detecting the presence of a single noncovalent interaction [a hydrogen bond (H-bond)] within a large DNA duplex in solution and measure its formation enthalpy (ΔHf). We report that introduction of a H-bond into the TC2═O group from the noncanonical nucleobase 2-aminopurine produces an expected decrease ∼10 ± 0.76 cm-1 (from ∼1720 cm-1 in Watson-Crick to ∼1710 cm-1 in 2-aminopurine), which correlates with an enthalpy of ∼0.93 ± 0.066 kcal/mol for this interaction.

Three-dimensional chitinous scaffolds often used in regenerative medicine, tissue engineering, biomimetics and technology are mostly isolated from marine organisms, such as marine sponges (Porifera). In this work, we report the results of the electrochemical isolation of the ready to use chitinous matrices from three species of verongiid demosponges (Aplysina archeri, Ianthella basta and Suberea clavata) as a perfect example of possible morphological and chemical dimorphism in the case of the marine chitin sources. The electrolysis of concentrated Na2SO4 aqueous solution showed its superiority over the chemical chitin isolation method in terms of the treatment time reduction: only 5.5 h for A. archeri, 16.5 h for I. basta and 20 h for the S. clavata sample. Further investigation of the isolated scaffolds by digital microscopy and SEM showed that the electrolysis-supported isolation process obtains chitinous scaffolds with well-preserved spatial structure and it can be competitive to other alternative chitin isolation techniques that use external accelerating factors such as microwave irradiation or atmospheric plasma. Moreover, the infrared spectroscopy (ATR-FTIR) proved that with the applied electrochemical conditions, the transformation into chitosan does not take place.

SAM (S‐adenosyl‐L‐Methionine) is the universal methyl donor used by cells, and is also the second most used cofactor after ATP. Methyltransferases catalyze the methylation of numerous biomolecules including DNA, protein, and RNA; and have a broad array of functions ranging from cell signaling to gene transcription. Abnormal levels of methyltransferases have been linked to malignant cellular states, cardiovascular diseases, and neurological diseases. Therefore, methyltransferases are an attractive drug target. Several inhibitors of histone PMTs (protein methyltransferases) have been used in the clinic, signifying PMTs as a target class. The SAM binding pocket in protein methyltransferases has been utilized by in drug design strategies. Yet, there is not high specificity when it comes to these drugs, making the drugs imperfect. Recent NMR work reveals that unbound SAM has high populations of different conformations in dynamic equilibrium, which means there may be multiple conformations when SAM is bound to methyltransferases. Having multiple conformations of SAM may allow for specific drugs to be designed to fit the unique pockets, allowing for higher drug specificity. Here we use vibrational spectroscopy, which is sensitive to molecular conformation and immune to timescale, to determine if these conformational dynamics persist when SAM is bound to PMTs. Molecular dynamic studies and small molecules studies support the hypothesis that various SAM conformations exist both when free and when bound to the proteins.

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)(4)-Cys(5-6) cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O-2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu(II) to form Cu(I)(4)Zn(II)(4)MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430-450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu(II) leads to the formation of a redox-inert Cu(I)(4)-Cys(5) cluster with short Cu-Cu distances (<2.8 angstrom), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu(II) reduction, generating the final Cu(I)(4)-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu(I), potentially explaining the observed DRA stability in the presence of O-2. We thus provide evidence that the MT-3 Cu(I)(4)-Cys(5) cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.

Supramolecular interactions provide 3D printable materials with noncovalent cross-linking and stimuli-responsive properties to improve their processability and functionality. Here, we developed photoprintable acrylate polymer networks that contain pendant aliphatic or aromatic urea groups at different molar ratios to improve their toughness and enable self-healing ability via hydrogen bonding. We synthesized two methacrylate monomers containing either an aliphatic or an aromatic urea motif, and we formulated them in liquid photoreactive resins. We 3D printed these formulations and evaluated their tensile mechanical strength, elongation, toughness, and self-healing properties. By increasing the supramolecular cross-linking density provided by the urea groups, we improved the mechanical strength up to 119%, and the toughness up to 205%, compared to the control, in formulations with 10% molar ratio of the aromatic urea monomer, without sacrificing the elongation of the printed parts. Physical evidence for the presence of hydrogen bonding was provided with variable temperature Fourier transform infrared (VT-ATR-FTIR) spectroscopy and van't Hoff analysis. The self-healing efficiency of these formulations was characterized by measuring the recovery of their tensile mechanical properties. Resins that contained 10 mol % of the aliphatic or aromatic urea monomers recovered more than 100% of their original mechanical strength. These results show the ability of supramolecular cross-linking via urea hydrogen bonding in improving the toughness, tuning the mechanical properties, and imparting stimuli-responsiveness in materials that are compatible with photoprinting methods.

Measuring the strength of the hydrogen bonds between DNA base pairs is of vital importance for understanding how our genetic code is physically accessed and recognized in cells, particularly during replication and transcription. Therefore, it is important to develop probes for these key hydrogen bonds (H-bonds) that dictate events critical to cellular function, such as the localized melting of DNA. The vibrations of carbonyl bonds are well-known probes of their H-bonding environment, and their signals can be observed with infrared (IR) spectroscopy. Yet, pinpointing a single bond of interest in the complex IR spectrum of DNA is challenging due to the large number of carbonyl signals that overlap with each other. Here, we develop a method using isotope editing and infrared (IR) spectroscopy to isolate IR signals from the thymine (T) C2=O carbonyl. We use solvatochromatic studies to show that the TC2=O signal's position in the IR spectrum is sensitive to the H-bonding capacity of the solvent. Our results indicate that C2=O of a single T base within DNA duplexes experiences weak H-bonding interactions. This finding is consistent with the existence of a third, noncanonical CH center dot center dot center dot O H-bond between adenine and thymine in both Watson-Crick and Hoogsteen base pairs in DNA.

Marine sponges were among the first multicellular organisms on our planet and have survived to this day thanks to their unique mechanisms of chemical defense and the specific design of their skeletons, which have been optimized over millions of years of evolution to effectively inhabit the aquatic environment. In this work, we carried out studies to elucidate the nature and nanostructural organization of three-dimensional skeletal microfibers of the giant marine demosponge Ianthella basta, the body of which is a micro-reticular, durable structure that determines the ideal filtration function of this organism. For the first time, using the battery of analytical tools including three-dimensional micro-X-ray Fluorescence (3D-µXRF), X-ray diffraction (XRD), infra-red (FTIR), Raman and Near Edge X-ray Fine Structure (NEXAFS) spectroscopy, we have shown that biomineral calcite is responsible for nano-tuning the skeletal fibers of this sponge species. This is the first report on the presence of a calcitic mineral phase in representatives of verongiid sponges which belong to the class Demospongiae. Our experimental data suggest a possible role for structural amino polysaccharide chitin as a template for calcification. Our study suggests further experiments to elucidate both the origin of calcium carbonate inside the skeleton of this sponge and the mechanisms of biomineralization in the surface layers of chitin microfibers saturated with bromotyrosines, which have effective antimicrobial properties and are responsible for the chemical defense of this organism. The discovery of the calcified phase in the chitinous template of I. basta skeleton is expected to broaden the knowledge in biomineralization science where the calcium carbonate is regarded as a valuable material for applications in biomedicine, environmental science, and even in civil engineering.

Selected hypermineralized bones (rostrum and tympanic bullae) have yet to be characterized for diverse species of whales (Cetacea). Hypermineralization in these structures is an example of extreme biomineralization that, however, occurs at temperatures around 36 °C. In this study we present the results of analytical investigations of the specimen of tympanic bulla isolated from gray whale ( Eschrichtius robustus ) for the first time. Examination of the internal surface of the bone mechanically crushed under a press revealed the presence of a lipid-containing phase, which did not disappear even after complete demineralization of the bone material. Additionally, analytical investigations including CARS, ATR-FTIR, Raman and XRD confirmed the presence of carbonated bioapatite and a collagen- lipid complex as the main components of this up to 2.34 kg/cm 3 dense bone. Our experimental results open the way for further research on understanding of the principles of hypermineralization in highly specialized whale bones.

Structure-based tissue engineering requires large-scale 3D cell/tissue manufacture technologies, to produce biologically active scaffolds. Special attention is currently paid to naturally pre-designed scaffolds found in skeletons of marine sponges, which represent a renewable resource of biomaterials. Here, an innovative approach to the production of mineralized scaffolds of natural origin is proposed. For the first time, a method to obtain calcium carbonate deposition ex vivo, using living mollusks hemolymph and a marine-sponge-derived template, is specifically described. For this purpose, the marine sponge Aplysin aarcheri and the terrestrial snail Cornu aspersum were selected as appropriate 3D chitinous scaffold and as hemolymph donor, respectively. The formation of calcium-based phase on the surface of chitinous matrix after its immersion into hemolymph was confirmed by Alizarin Red staining. A direct role of mollusks hemocytes is proposed in the creation of fine-tuned microenvironment necessary for calcification ex vivo. The X-ray diffraction pattern of the sample showed a high CaCO3 amorphous content. Raman spectroscopy evidenced also a crystalline component, with spectra corresponding to biogenic calcite. This study resulted in the development of a new biomimetic product based on ex vivo synthetized ACC and calcite tightly bound to the surface of 3D sponge chitin structure.

Marine demosponges of the Verongiida order are considered a gold-mine for bioinspired materials science and marine pharmacology. The aim of this work was to simultaneously isolate selected bromotyrosines and unique chitinous structures from A. aerophoba and to propose these molecules and biomaterials for possible application as antibacterial and antitumor compounds and as ready-to-use scaffolds for cultivation of cardiomyocytes, respectively. Among the extracted bromotyrosines, the attention has been focused on aeroplysinin-1 that showed interesting unexpected growth inhibition properties for some Gram-negative clinical multi-resistant bacterial strains, such as A. baumannii and K. pneumoniae, and on aeroplysinin-1 and on isofistularin-3 for their anti-tumorigenic activity. For both compounds, the effects are cell line dependent, with significant growth inhibition activity on the neuroblastoma cell line SH-SY5Y by aeroplysinin-1 and on breast cancer cell line MCF-7 by isofistularin-3. In this study, we also compared the cultivation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) on the A. aerophoba chitinous scaffolds, in comparison to chitin structures that were pre-coated with Geltrex™, an extracellular matrix mimetic which is used to enhance iPSC-CM adhesion. The iPSC-CMs on uncoated and pure chitin structures started contracting 24 h after seeding, with comparable behaviour observed on Geltrex-coated cell culture plates, confirming the biocompatibility of the sponge biomaterial with this cell type. The advantage of A. aerophoba is that this source organism does not need to be collected in large quantities to supply the necessary amount for further pre-clinical studies before chemical synthesis of the active compounds will be available. A preliminary analysis of marine sponge bioeconomy as a perspective direction for application of biomaterials and secondary bioactive metabolites has been finally performed for the first time.

Molluscan shells are an example of a mineral-based biocomposite material, and most studies to date have focused on understanding their biomineralization mechanisms. Meanwhile, large amounts of these shells are produced as waste globally by seafood which is used by other industries as a source of biogenic calcium carbonates. In this study, we propose a simple methodological approach for isolation of Conchixes , the organic scaffolds that resemble the size and shapes of mollusks shells, using gentle EDTA-based demineralization of the shells. Such mineral-free biological materials have been extracted from selected representatives of marine and fresh water bivalves, as well as from marine and terrestrial gastropods under study. Key pathways to practical applications of molluscan conchixes with regards to pharmacy, cosmetics, feed and feed additives, biomedicine and bioinspired materials science are also discussed.

A( syn)-T and G( syn)-C+ Hoogsteen base pairs in protein-bound DNA duplexes can be difficult to resolve by X-ray crystallography due to ambiguous electron density and by nuclear magnetic resonance (NMR) spectroscopy due to poor chemical shift dispersion and size limitations with solution-state NMR spectroscopy. Here we describe an NMR strategy for characterizing Hoogsteen base pairs in protein-DNA complexes, which relies on site-specifically incorporating 13C- and 15N-labeled nucleotides into DNA duplexes for unambiguous resonance assignment and to improve spectral resolution. The approach was used to resolve the conformation of an A-T base pair in a crystal structure of an ∼43 kDa complex between a 34 bp duplex DNA and the integration host factor (IHF) protein. In the crystal structure (Protein Data Bank entry 1IHF ), this base pair adopts an unusual Hoogsteen conformation with a distorted sugar backbone that is accommodated by a nearby nick used to aid in crystallization. The NMR chemical shifts and interproton nuclear Overhauser effects indicate that this base pair predominantly adopts a Watson-Crick conformation in the intact DNA-IHF complex under solution conditions. Consistent with these NMR findings, substitution of 7-deazaadenine at this base pair resulted in only a small (∼2-fold) decrease in the IHF-DNA binding affinity. The NMR strategy provides a new approach for resolving crystallographic ambiguity and more generally for studying the structure and dynamics of protein-DNA complexes in solution.