Cations as Mediators of the Adsorption of Nucleic Acids on Clay Surfaces in Prebiotic Environments

Monovalent ([Na⁺] > 10 mM) and divalent ([Ca²⁺], [Mg²⁺] > 1.0 mM) cations induced the precipitationof nucleic acid molecules. In the presence of clay minerals (montmorillonite and kaolinite), there was adsorption instead of precipitation. The cation concentration needed for adsorption depended on both the valence of the cations and the chemical nature of the nucleic acid molecules. Double-stranded nucleic acids needed higher cation concentrations than single-stranded ones to be adsorbed to the same extent on clay. Divalent cations were more efficient than monovalent ones in mediating adsorption. Adsorption to the clay occurred only when both nucleic acids and cations were present. However, once the complexes were formed, the cations could not be removed from the system by washing, indicating that they are directly involved in the association between nucleic acids and mineral surfaces.These observations indicate that cations take part directly in the formation of nucleic acid-clay complexes, acting as a `bridge' between the negative charges on the mineral surface and those of the phosphate groups of the genetic polymer. The relatively low cation concentrations needed for adsorption and the ubiquitous presence of clay minerals in the environment suggest that the adsorption of nucleic acids on mineral surfaces could have taken place in prebiotic habitats. This may have played an important role in the formation and preservation of nucleic acids and/or their precursor polymers.     

Mineral Surface Directed Membrane Assembly

The transition from non-living to living matter may have resulted from the self-organizing properties of organic molecules and their interactions with a chemically rich inorganic environment. We have shown that a solution containing RNA, fatty acids and clay produces structures that contain a potentially catalytic surface (clay) and a potential informational biopolymer (RNA) encapsulated within a membrane. This highlights the ability of mineral surfaces to bring together and organize key components of primordial life. We have extended our analysis of mineral-mediated vesicle catalysis to include other natural minerals and synthetic surfaces of varying shape, size, and charge density. Our results show that while RNA polymerization on minerals may be restricted to the surface environment provided by montmorillonite, vesicle formation is enhanced in the presence of disparate types of surfaces. A model is presented in which new sheets of amphiphiles form just proximal to a surface. Similar interactions between amphiphiles and minerals on early Earth may have resulted in the encapsulation of a diverse array of mineral particulates with catalytic properties.     

Invertible micellar polymer assemblies for delivery of poorly water-soluble drugs

Strategically designed amphiphilic invertible polymers (AIPs) are capable of (i) self-assembling into invertible micellar assemblies (IMAs) in response to changes in polarity of environment, polymer concentration, and structure, (ii) accommodating (solubilizing) substances that are otherwise insoluble in water, and (iii) inverting their molecular conformation in response to changes in the polarity of the local environment. The unique ability of AIPs to invert the molecular conformation depending on the polarity of the environment can be a decisive factor in establishing the novel stimuli-responsive mechanism of solubilized drug release that is induced just in response to a change in the polarity of the environment. The IMA capability to solubilize lipophilic drugs and deliver and release the cargo molecules by conformational inversion of polymer macromolecules in response to a change of the polarity of the environment was demonstrated by loading IMA with a phytochemical drug, curcumin. It was demonstrated that four sets of micellar vehicles based on different AIPs were capable of delivering the curcumin from water to an organic medium (1-octanol) by means of unique mechanism: AIP conformational inversion in response to changing polarity from polar to nonpolar. The IMAs are shown to be nontoxic against human cells up to a concentration of 10 mg/L. On the other hand, the curcumin-loaded IMAs are cytotoxic to breast carcinoma cells at this concentration, which confirms the potential of IMA-based vehicles in controlled delivery of poorly water-soluble drug candidates and release by means of this novel stimuli-responsive mechanism.     

Formation of vesicles through solvent assisted self-assembly of hydrophobic pentapeptides: encapsulation and pH responsive release of dyes by the vesicles

In the biomimetic design two hydrophobic pentapetides Boc-Ile-Aib-Leu-Phe-Ala-OMe (I) and Boc-Gly-Ile-Aib-Leu-Phe-OMe (II) (Aib: α-aminoisobutyric acid) containing one Aib each are found to undergo solvent assisted self-assembly in methanol/water to form vesicular structures, which can be disrupted by simple addition of acid. The nanovesicles are found to encapsulate dye molecules that can be released by the addition of acid as confirmed by fluorescence microscopy and UV studies. The influence of solvent polarity on the morphology of the materials generated from the peptides has been examined systematically, and shows that fibrillar structures are formed in less polar chloroform/petroleum ether mixture and vesicular structures are formed in more polar methanol/water. Single crystal X-ray diffraction studies reveal that while β-sheet mediated self-assembly leads to the formation of fibrillar structures, the solvated β-sheet structure leads to the formation of vesicular structures. The results demonstrate that even hydrophobic peptides can generate vesicular structures from polar solvent which may be employed in model studies of complex biological phenomena.     

Comparative theoretical study of the conformations of amphotericin methyl ester and amphotericin B polar heads in the presence of water.

Amphotericin methyl ester (AmE) is an interesting derivative of amphotericin B (AmB) because of its enhancement of selectivity against the fungicells. Both AmB and AmE molecules differ by the structure of their polar heads. This work deals with a theoretical study of conformations of the polar head of AmE in the presence of hydration water molecules. The results will be compared with our previous work concerning AmB.     

Incorporation of NSAIDs in micelles: implication of structural switchover in drug-membrane interaction

Non-steroidal anti-inflammatory drugs (NSAIDs) of oxicam group are not only effective as anti-inflammatory agents but also show diverse functions. Their principal targets are cyclooxygenases, which are membrane-associated enzymes. To bind with the targets these drugs have to pass through the membrane and hence their interactions with biomembranes should play a major role in guiding their interactions with cyclooxygenases. Here we have studied the interactions of three NSAIDs of oxicam group viz. piroxicam, meloxicam and tenoxicam with micelles having different headgroup charges, as simple membrane mimetic systems. Spectroscopic methods have been used to understand the interaction of these drugs with Cetyl N,N,N-trimethyl ammonium bromide (cationic), Sodium dodecyl sulphonate (anionic) and Triton X-100 (neutral) micelles. Our results demonstrate that the environment of the drugs i.e. the nature of the micelles plays a decisive role in choosing a specific prototropic form of the drugs for incorporation. Additionally it induces a switch over or change between different prototropic forms of piroxicam, which is correlated with the change in their reactivities in presence of different surface charges, given by the change in pK(a) values. These results together, indicate that in vivo, the diverse nature of biomembranes might play a significant role in choosing the particular form of oxicam NSAIDs that would be presented to their targets.     

Interaction of piroxicam and meloxicam with DMPG/DMPC mixed vesicles: anomalous partitioning behavior

Small unilamellar vesicles (SUVs) formed from a mixture of dimyristoylphosphatidylcholine (zwitterionic lipid with bulkier headgroup) and dimyristoylphosphatidylglycerol (anionic lipid with relatively smaller headgroup) allows better modulation of the physical properties of lipid bilayers compared to SUVs formed by a single type of lipid, providing us with a better model system to study the effect of membrane parameters on the partitioning of small molecules. Membrane parameter like packing of the vesicles is more pronounced in the gel phase and hence the study was carried out in the gel phase. Mixed vesicles formed from DMPG and DMPC with the mole percent ratio of 100:0, 90:10 and 80:20 were used for this study. As examples of polar solutes, piroxicam and meloxicam, two Non Steroidal Anti-inflammatory Drugs (NSAIDs) were chosen. The pH was adjusted to 2.8 in order to eliminate the presence of anionic forms of the drugs that would not approach the vesicles containing negatively charged DMPG (50% deprotonated at pH 2.8). Surface potential measured by using TNS (2,6-p-toluidinonaphthalene sulfonate, sodium salt) as surface charge sensitive probe showed no significant changes in the surface electrostatics in increasing DMPC content from 0 to 20%. Transmission electron microscopy (TEM) was used to characterize SUVs of different composition at pH 2.8. The average diameter of the mixed vesicles was found to be smaller than that formed by DMPG and DMPC alone. Partition coefficient (K(P)) of piroxicam and meloxicam was measured using intrinsic fluorescence of these molecules. K(P) value of piroxicam decreases with increase in DMPC content whereas it increases with DMPC content in case of meloxicam. This anomalous behavior of partitioning is unexpected since there was no significant change in surface pH of the vesicles and has been explained in terms of lipid packing and water penetration in the lipid bilayer.     

Deducing hydration sites of a protein from molecular dynamics simulations

Invariant water molecules that are of structural or functional importance to proteins are detected from their presence in the same location in different crystal structures of the same protein or closely related proteins. In this study we have investigated the location of invariant water molecules from MD simulations of ribonuclease A, HIV1-protease and Hen egg white lysozyme. Snapshots of MD trajectories represent the structure of a dynamic protein molecule in a solvated environment as opposed to the static picture provided by crystallography. The MD results are compared to an analysis on crystal structures. A good correlation is observed between the two methods with more than half the hydration sites identified as invariant from crystal structures featuring as invariant in the MD simulations which include most of the functionally or structurally important residues. It is also seen that the propensities of occupying the various hydration sites on a protein for structures obtained from MD and crystallographic studies are different. In general MD simulations can be used to predict invariant hydration sites when there is a paucity of crystallographic data or to complement crystallographic results.     

Solvent effect on cation–π interactions with Al3+

Cation-π interactions are known to be one of the strongest noncovalent forces in the gas phase, but they rarely occur in a fully solvated environment. The present work used two different ab initio molecular dynamics-based approaches to describe the correlation between the strength of the cation-π interactions and the number of water molecules surrounding the cation. Five different complexes between an aluminum cation and different molecules containing aromatic rings were studied, and the degree of hydration of each complex was varied. Results indicated that cation-π interactions vanish when the aluminum cation is surrounded by more than three water molecules. The results also highlighted the influence of -OH ligands on the interaction strength.     

Looking for the Primordial Genetic Honeycomb

All life forms on Earth share the same biological program based on the DNA/RNA genomes and proteins. The genetic information, recorded in the nucleotide sequence of the DNA and RNA molecule, supplies the language of life which is transferred through the different generations, thus ensuring the perpetuation of genetic information on Earth. The presence of a genetic system is absolutely essential to life. Thus, the appearance in an ancestral era of a nucleic acid-like polymer able to undergo Darwinian evolution indicates the beginning of life on our planet. The building of primordial genetic molecules, whatever they were, required the presence of a protected environment, allowing the synthesis and concentration of precursors (nucleotides), their joining into larger molecules (polynucleotides), the protection of forming polymers against degradation (i.e. by cosmic and UV radiation), thus ensuring their persistence in a changing environment, and the expression of the “biological” potential of the molecule (its capacity to self-replicate and evolve). Determining how these steps occurred and how the primordial genetic molecules originated on Earth is a very difficult problem that still must be resolved. It has long been proposed that surface chemistry, i.e. on clay minerals, could have played a crucial role in the prebiotic formation of molecules basic to life. In the present work, we discuss results obtained in different fields that strengthen the hypothesis of a clay-surface-mediated origin of genetic material. Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

An exploratory theoretical elucidation on the peroxyl-radical-scavenging mechanism and structure-activity relationship of nonsteroidal anti-inflammatory drugs

The peroxyl-radical-scavenging mechanism of some nonsteroidal anti-inflammatory drugs (NSAIDs), namely tolmetin, ketorolac, indomethacin, acemetacin, and oxaprozin, is clarified by combined density functional theory (DFT) calculations. It is revealed that H-atom-abstraction rather than electron transfer reaction is involved in the radical-scavenging process of these NSAIDs in polar aqueous solution. This seems contrary to the common viewpoint that the latter is predominant in polar media. The calculated results also show that H-atom at C(beta) or C(gamma) position is readily to be abstracted, and the lowest C-H bond dissociation enthalpy (BDE) can qualitatively account for the activity difference for the five NSAIDs.     

Hydration of B-DNA: comparison between the water network around poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) on the basis of Monte Carlo computations

A computational method is elaborated for studying the water environment around regular polynucleotide duplexes; it allows rigorous structural information on the hydration shell of DNA to be obtained. The crucial aspect of this Monte Carlo simulation is the use of periodical boundary conditions. The output data consists of local maxima of water density in the space near the DNA molecule and the properties of one- and two-membered water bridges as function of pairs of polar groups of DNA. In the present paper the results for poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) are presented. The differences in their hydration shells are of a purely structural nature and are caused by the symmetry of the polar groups of the polymers under study, the symmetry being reflected by the hydration shell. The homopolymer duplex hydration shell mirrors the mononucleotide repeat. The water molecules contacting the polynucleotide in the minor groove are located nearly in the plane midway between the planes of successive base pairs. One water molecule per base pair forms a water bridge facing two polar groups of bases from adjacent base pairs and on different strands making a "spine"-like structure. In contrast, the major groove hydration is stabilized exclusively by two-membered water bridges; the water molecules deepest in the groove are concentrated near the plane of the corresponding base pair. The alternating polymer is characterized by a marked dyad symmetry of the hydration shell corresponding to the axis between two successive base pairs. The minor groove hydration of the dCpdG step resembles the characteristic features of the homopolymer, but the bridge between the O2 oxygens of the other base-stacking type is formed by two water molecules. The major groove hydration is characterized by high probability of one-membered water bridges and by localization of a water molecule on the dyad axis of the dGpdC step. The found structural elements are discussed as reasonable invariants of a dynamic hydration shell.     

Solvated docking: introducing water into the modelling of biomolecular complexes

MOTIVATION: Interfacial water, which plays an important role in mediating biomolecular interactions, has been neglected in the modelling of biomolecular complexes. METHODS: We present a solvated docking approach that explicitly accounts for the presence of water in protein-protein complexes. Our solvated docking protocol is based on the concept of the first encounter complex in which a water layer is present in-between the molecules. It mimics the pathway from this initial complex towards the final assembly in which most waters have been expelled from the interface. Docking is performed from solvated biomolecules and waters are removed in a biased Monte Carlo procedure based on water-mediated contact propensities obtained from an analysis of high-resolution crystal structures. RESULTS: We demonstrate the feasibility of this approach for protein-protein complexes representing both 'wet' and 'dry' interfaces. Solvated docking leads to improvements both in quality and scoring. Water molecules are recovered that closely match the ones in the crystal structures. AVAILABILTY: Solvated docking will be made available in the future release of HADDOCK version 2.0 (http://www.nmr.chem.uu.nl/haddock).     

Montmorillonite protection of an UV-irradiated hairpin ribozyme: evolution of the RNA world in a mineral environment

Background

The hypothesis of an RNA-based origin of life, known as the "RNA world", is strongly affected by the hostile environmental conditions probably present in the early Earth. In particular, strong UV and X-ray radiations could have been a major obstacle to the formation and evolution of the first biomolecules. In 1951, J. D. Bernal first proposed that clay minerals could have served as the sites of accumulation and protection from degradation of the first biopolymers, providing the right physical setting for the evolution of more complex systems. Numerous subsequent experimental studies have reinforced this hypothesis.

Results

The ability of the possibly widespread prebiotic, clay mineral montmorillonite to protect the catalytic RNA molecule ADHR1 (Adenine Dependent Hairpin Ribozyme 1) from UV-induced damages was experimentally checked. In particular, the self-cleavage reaction of the ribozyme was evaluated after UV-irradiation of the molecule in the absence or presence of clay particles. Results obtained showed a three-fold retention of the self-cleavage activity of the montmorillonite-protected molecule, with respect to the same reaction performed by the ribozyme irradiated in the absence of the clay.

Conclusion

These results provide a suggestion with which RNA, or RNA-like molecules, could have overcame the problem of protection from UV irradiation in the RNA world era, and suggest that a clay-rich environment could have favoured not only the formation of first genetic molecules, but also their evolution towards increasingly complex molecular organization.

     

Effect of Different Antibiotics and Non-Steroidal Anti-Inflammatory Drugs on the Growth of Lactobacillus casei Shirota

The purpose of this study was to investigate whether some non-steroidal anti-inflammatory drugs (NSAIDs) could cause inhibition of the growth of Lactobacillus casei Shirota (LcS) or whether this microorganism is able to use some of them as the sole carbon source, considering that the simultaneous consumption of NSAIDs and a dairy drink fermented with LcS could help to prevent the appearance or improve the healing of gastric ulcers. Acetylsalicylic acid (ASA), sodium acetylsalicylate (SAS), acetaminophen, sodium naproxen, and sodium ibuprofen were added as the sole carbon source to a basal medium and tested for biodegradation by LcS. The same NSAIDs were added in different concentrations to disks and plated on MRS Agar to test the possible inhibitory effect of these compounds on LcS. Also, the resistance of LcS to 12 different antibiotics was studied on MRS agar. None of the NSAIDs tested could be used by LcS as the sole carbon source at the assayed concentrations. In the case of the disk diffusion method, sodium naproxen showed inhibition zones for the 500-μg disks and sodium ibuprofen was inhibitory for the 250- and 500-μg disks. However, when the macrobroth dilution method was used, the growth of LcS was inhibited by ASA, SAS, acetaminophen, and sodium ibuprofen. This strain showed resistance to the antibiotics sulfamethoxazole with trimethoprim, pefloxacin, and gentamicin. This is the first study on the effect of NSAIDs on probiotic bacteria. The results of the biodegradation test indicate that the simultaneous consumption of NSAIDs and a dairy beverage with LcS is not likely to change the bioavailability of the drugs.     

Effects of non-steroid anti-inflammatory drugs in membrane bilayers

The thermal effects of non-steroidal anti-inflammatory drugs (NSAIDs) meloxicam, tenoxicam, piroxicam and lornoxicam have been studied in dipalmitoylphosphatidylcholine (DPPC) membrane bilayers using neutral and acidic environments (pH 2.5). The strength of the perturbing effect of the drugs is summarized to a lowering of the main phase transition temperature and a broadening of the phase transition temperature as well as broadening or abolishment of the pretransition of DPPC bilayers. The thermal profiles in the two environments were very similar. Among the NSAIDs studied meloxicam showed the least perturbing effect. The differential scanning calorimetry results (DSC) in combination with molecular modeling studies point out that NSAIDs are characterized by amphoteric interactions and are extended between the polar and hydrophobic segments of lipid bilayers. The effects of NSAIDs in membrane bilayers were also investigated using Raman spectroscopy. Meloxicam showed a gauche:trans profile similar to DPPC bilayers while the other NSAIDs increased significantly the gauche:trans ratio. In conclusion, both techniques show that in spite of the close structural similarity of the NSAIDs studied, meloxicam appears to have the lowest membrane perturbing effects probably attributed to its highest lipophilicity.     

Why do water molecules around small hydrophobic solutes form stronger hydrogen bonds than in the bulk?

Molecular solutes are known to have a strong effect on the structural and dynamical properties of the surrounding water. In our recent study (PNAS, 114, 322 (2017)) we have identified the presence of strengthened water hydrogen bonds near hydrophobic solutes by using both IR spectroscopy and ab-initio molecular dynamics simulations. The water molecules involved in the enhanced hydrogen bonding have been shown to display extensive structural ordering and restricted mobility. We observed that an individual pair of water molecules can make stronger hydrogen bond to each other if it is not surrounded by intercalating water molecules. Here we present compelling simulation results which unravel a simple mechanistic picture of the emergence of the hydrogen bond (HB) strengthening around solvated methane. We show explicitly that actual absence of water molecules within the excluded volume due to the hydrophobic molecule assures smaller residual torque on neighboring water molecules enabling the formation of stronger HBs between them.     

Molecular Modelling of The Mechanism of Action of Organic Clay-Swelling Inhibitors

Abstract

It is well known that the sodium smectite class of clays swells macroscopically in contact with water, whereas under normal conditions the potassium form does not. In recent work using molecular simulation methods, we have provided a quantitative explanation both for the swelling behaviour of sodium smectite clays and the lack of swelling of potassium smectites [1]. In the present paper, we apply similar modelling methods to study the mechanism of inhibition of clay-swelling by a range of organic molecules.

Experimentally, it is known that polyalkylene glycols (polyethers) of intermediate to high relative molecular mass are effective inhibitors of smectite clay swelling. We use a range of atomistic simulation techniques, including Monte Carlo and molecular dynamics, to investigate the interactions between a selection of these compounds, water, and a model smectite clay mineral. These interactions occur by means of organised intercalation of water and organic molecules within the galleries between individual clay layers.

The atomic interaction potentials deployed in this work are not as highly optimised as those used in our clay-cation-water work [1]. Nevertheless, our simulations yield trends and results that are in qualitative and sometimes semi-quantitative agreement with experimental findings on similiar (but not identical) systems. The internal energy of adsorption of simple polyethers per unit mass on the model clay is not significantly different from that for water adsorption; our Monte Carlo studies indicate that entropy is the driving force for the sorption of the simpler organic molecules inside the clay layers: a single long chain polyethylene glycol can displace a large number of water molecules, each of whose translational entropy is greatly enhanced when outside the clay. Hydrophobically modified polyalkylene glycols also enjoy significant van der Waals interactions within the layers which they form within the clay galleries.

In conjunction with experimental studies, our work furnishes valuable insights into the relative effectiveness of the compounds considered and reveals the generic features that high performance clay-swelling inhibitors should possess. For optimal inhibitory activity, these compounds should be reasonably long chain linear organic molecules with localised hydrophobic and hydrophilic regions along the chain. On intercalation of these molecules within the clay layers, the hydrophobic regions provide an effective seal against ingress of water, while the hydrophilic ones enhance the binding of the sodium cations to the clay surface, preventing their hydration and the ensuing clay swelling.     

Interaction of poly(α-L-glutamic acid) and sodium poly(α-L-glutamate) with solvent components in water–2-chloroethanol mixtures

The preferential interaction of sodium poly(α-L -glutamate) and poly(α-L -glutamic acid) with the solvent components in water/2-chloroethanol mixtures has been determined using density-increment measurements. The degree of preferential interaction was deduced from the density increments at constant molality of 2-chloroethanol and at constant chemical potential of 2-chloroethanol. Sodium poly(α-L -glutamate) and poly(α-L -glutamic acid) are both preferentially hydrated in the whole range of solvent composition. A dehydration process occurs during the 2-chloroethanol-induced coil-to-helix transition of sodium poly(α-L -glutamate). This dehydration process was attributed to the release of some moles of water from the neighborhood of the peptide bond during the nucleation of the helix. After the conformational transition, sodium poly(α-L -glutamate) is solvated by one 2-chloroethanol molecule. The location of water and 2-chloroethanol molecules in the different parts of the residue (more polar and less polar portions) is also discussed.