Liquid-Liquid Phase Separation Mediated Formation of Chiral 2D Crystalline Nanosheets of a Co-Assembled System.

The role of macromolecule-macromolecule and macromolecule-H2O interactions and the resulting perturbation of the H-bonded network of H2O in the liquid-liquid phase separation (LLPS) process of biopolymers are well-known. However, the potential of the hydrated state of supramolecular structures (non-covalent analogs of macromolecules) of synthetic molecules is not widely recognized for playing a similar role in the LLPS process. Herein, LLPS occurred during the co-assembly of hydrated supramolecular vesicles (bolaamphiphile, BA1) with a net positive charge (zeta potential, ζ = +60 ± 2 mV) and a dianionic chiral molecule (disodium l-[+]-tartrate) is reported. As inferred from cryo-transmission electron microscopy (TEM), the LLPS-formed droplets serve as the nucleation precursors, dictating the structure and properties of the co-assembly. The co-assembled structure formed by LLPS effectively integrates the counter anion's asymmetry, resulting in the formation of ultrathin free-standing, chiral 2D crystalline sheets. The significance of the hydrated state of supramolecular structures in influencing LLPS is unraveled through studies extended to a less hydrated supramolecular structure of a comparable system (BA2). The role of LLPS in modulating the hydrophobic interaction in water paves the way for the creation of advanced functional materials in an aqueous environment.

A Minor Groove Binder with Significant Cytotoxicity on Human Lung Cancer Cells: The Potential of Hesperetin Functionalised Silver Nanoparticles.

Natural drug functionalised silver (Ag) nanoparticles (NPs) have gained significant interest in pharmacology related applications due to their therapeutic efficiency. We have synthesised silver nanoparticle using hesperetin as a reducing and capping agent. This work aims to discuss the relevance of the hesperetin functionalised silver nanoparticles (H-AgNPs) in the field of nano-medicine. The article primarily investigates the anticancer activity of H-AgNPs and then their interactions with calf thymus DNA (ctDNA) through spectroscopic and thermodynamic techniques. The green synthesised H-AgNPs are stable, spherical in shape and size of 10 ± 3 nm average diameter. The complex formation of H-AgNPs with ctDNA was established by UV-Visible absorption, fluorescent dye displacement assay, isothermal calorimetry and viscosity measurements. The binding constants obtained from these experiments were consistently in the order of 104 Mol-1. The melting temperature analysis and FTIR measurements confirmed that the structural alterations of ctDNA by the presence of H-AgNPs are minimal. All the thermodynamic variables and the endothermic binding nature were acquired from ITC experiments. All these experimental outcomes reveal the formation of H-AgNPs-ctDNA complex, and the results consistently verify the minor groove binding mode of H-AgNPs. The binding constant and limit of detection of 1.8 µM found from the interaction studies imply the DNA detection efficiency of H-AgNPs. The cytotoxicity of H-AgNPs against A549 and L929 cell lines were determined by in vitro MTT cell viability assay and lactate dehydrogenase (LDH) assay. The cell viability and LDH enzyme release are confirmed that the H-AgNPs has high anticancer activity. Moreover, the calculated LD50 value for H-AgNPs against lung cancer cells is 118.49 µl/ml, which is a low value comparing with the value for fibroblast cells (269.35 µl/ml). In short, the results of in vitro cytotoxicity assays revealed that the synthesised nanoparticles can be considered in applications related to cancer treatments. Also, we have found that, H-AgNPs is a minor groove binder, and having high DNA detection efficiency.

Exploring the cytotoxicity on human lung cancer cells and DNA binding stratagem of camptothecin functionalised silver nanoparticles through multi-spectroscopic, and calorimetric approach.

The influence of nanoparticles inside the human body and their interactions with biological macromolecules need to be explored/studied prior to specific applications. The objective of this study is to find the potential of camptothecin functionalised silver nanoparticles (CMT-AgNPs) in biomedical applications. This article primarily investigates the binding stratagem of CMT-AgNPs with calf thymus DNA (ctDNA) through a series of spectroscopic and calorimetric methods and then analyses the anticancer activity and cytotoxicity of CMT-AgNPs. The nanoparticles were synthesized using a simple one pot method and characterized using UV-Visible, fourier transform infrared (FTIR) spectroscopy, X-ray diffraction and high-resolution transmission electron microscopy (HRTEM). The average size of CMT-AgNPs is 10 ± 2 nm. A group of experimental techniques such as UV-Visible spectrophotometry, fluorescence dye displacement assay, circular dichroism (CD) and viscosity analysis unravelled the typical groove binding mode of CMT-AgNPs with ctDNA. The CD measurement evidenced the minor conformational alterations of double helical structure of ctDNA in the presence of CMT-AgNPs. The information deduced from the isothermal titration calorimetry (ITC) experiment is that the binding was exothermic and spontaneous in nature. Moreover, all the thermodynamic binding parameters were extracted from the ITC data. The binding constants obtained from UV absorption experiments, fluorescence dye displacement studies and ITC were consistently in the order of 104 Mol-1. All these results validated the formation of CMT-AgNPs-ctDNA complex and the results unambiguously confirm the typical groove binding mode of CMT-AgNPs. An exhaustive in vitro MTT assay by CMT-AgNPs and CMT against A549, HT29, HeLa and L929 cell lines revealed the capability of CMT-AgNPs as a potential anticancer agent.

Photophysical and thermodynamic evaluation on the in vitro and in silico binding profile of Camptothecin with DNA.

Camptothecin (CMT) is an anti-tumour alkaloid drug exhibiting selective topoisomerase-I inhibitory activity by eventually hindering dynamic functions of DNA duplex via initiating apoptosis. Unravelling the binding mechanism of CMT with bio macromolecular systems can offer fundamental information regarding the mechanism of actions which can lead to the design of rational proactive drugs. This study endeavoured the binding interactions of CMT with calf thymus DNA (ct-DNA) along with the structural alterations attained by the DNA duplex owing to CMT interactions through multi-spectroscopic, calorimetric and molecular docking studies. The UV-visible absorbance and fluorescence quenching studies revealed the binding strength of CMT with ct-DNA, evident from the binding constants K1 = 3.79 × 103 M-1 and Kq = 2 × 103 M-1. The time-resolved lifetime measurements inferred that the quenching was static due to the non-fluorescent ground state complex formation. The dye displacement study, temperature melting and viscosity measurements established a typical non-intercalative binding mode of CMT with ct-DNA. The binding isotherm deduced from ITC was found to be spontaneous and exothermic exerting a promising ΔG value of -6.2 kcal mol-1. The thermal kinetic parameters implied that the forces primarily involved in the CMT-ct-DNA complexation are hydrogen bonding and van der Waals interactions. Moreover, the structural alterations of DNA duplex reflected in the CD and FTIR spectra could undeniably confirm the groove binding manner of CMT. The in silico extra precision docking study explored more accurate molecular illustrations of sequence specific minor groove binding mechanism evolved between CMT and DNA corroborating well with the experimental results. These innovative findings may shorten the path towards the development of novel and more effective CMT drug derivatives.

Experimental Probing and Molecular Dynamics Simulation of the Molecular Recognition of DNA Duplexes by the Flavonoid Luteolin.

Luteolin (C15H10O6) is an important flavonoid found in many fruits, plants, medicinal herbs, and vegetables exhibiting many pharmacological properties. The anticancer, antitumor, antioxidant, and anti-inflammatory activities of luteolin have been reported. The pharmacological action of small molecules is dependent upon its interaction with biomacromolecules. The interactions of small molecules with DNA play a major role in the transcription and translation process. In this work, we explored the energetic profile of DNA-luteolin interaction by isothermal titration calorimetry (ITC). The effect of temperature and salt concentration on DNA binding was examined by UV-Vis method. The mode of interaction was further probed by UV melting temperature analysis and differential scanning calorimetry. An atomic level insight on the recognition of luteolin with DNA was achieved by employing molecular dynamics (MD) simulation on luteolin in complex with AT- and GC-rich DNA sequences. AMBER force field proves to be appropriate in providing an understanding on the binding mode and specificity of luteolin with duplex DNA. MD results suggest a minor groove binding of luteolin with DNA and the binding free energy obtained is in agreement with the experimental results.

A comprehensive approach to ascertain the binding mode of curcumin with DNA.

Curcumin is a natural phytochemical from the rhizoma of Curcuma longa, the popular Indian spice that exhibits a wide range of pharmacological properties like antioxidant, anticancer, anti-inflammatory, antitumor, and antiviral activities. In the published literatures we can see different studies and arguments on the interaction of curcumin with DNA. The intercalative binding, groove binding and no binding of curcumin with DNA were reported. In this context, we conducted a detailed study to understand the mechanism of recognition of dimethylsulfoxide-solubilized curcumin by DNA. The interaction of curcumin with calf thymus DNA (ctDNA) was confirmed by agarose gel electrophoresis. The nature of binding and energetics of interaction were studied by Isothermal Titration Calorimetry (ITC), Differential Scanning Calorimetry (DSC), UV-visible, fluorescence and melting temperature (Tm) analysis. The experimental data were compared with molecular modeling studies. Our investigation confirmed that dimethylsulfoxide-solubilized curcumin binds in the minor groove of the ctDNA without causing significant structural alteration to the DNA.

Conformational features of benzo-homologated yDNA duplexes by molecular dynamics simulation.

yDNA is a base-modified nucleic acid duplex containing size-expanded nucleobases. Base-modified nucleic acids could expand the genetic alphabet and thereby enhance the functional potential of DNA. Unrestrained 100 ns MD simulations were performed in explicit solvent on the yDNA NMR sequence [5'(yA T yA yA T yA T T yA T)2 ] and two modeled yDNA duplexes, [5'(yC yC G yC yC G G yC G G)2 ] and [(yT5' G yT A yC yG C yA yG T3')•(yA5' C T C yG C G yT A yC A3')]. The force field parameters for the yDNA bases were derived in consistent with the well-established AMBER force field. Our results show that DNA backbone can withstand the stretched size of the bases retaining the Watson-Crick base pairing in the duplexes. The duplexes retained their double helical structure throughout the simulations accommodating the strain due to expanded bases in the backbone torsion angles, sugar pucker and helical parameters. The effect of the benzo-expansion is clearly reflected in the extended C1'-C1' distances and enlarged groove widths. The size expanded base modification leads to reduction in base pair twist resulting in larger overlapping area between the stacked bases, enhancing inter and intra strand stacking interactions in yDNA in comparison with BDNA. This geometry could favour enhanced interactions with the groove binders and DNA binding proteins., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 55-64, 2016.

Energetics, Thermodynamics, and Molecular Recognition of Piperine with DNA.

Piperine, the bioactive phytochemical from black pepper (Piper nigrum L.), is a nontoxic natural compound exhibiting many physiological and pharmacological properties. They include antioxidant, anti-inflammatory, antimutagenic, antitumor, antiapoptotic, antigenotoxic, antiarthritic, antifungal, antimicrobial, antidepressant, anti-HBV, and gastro-protective activities. It also enhances the bioavailability of phytochemicals and drugs. The molecular mechanism of action of piperine with DNA has not yet been addressed, while its pharmacological activities have been reported. In this work we report for the first time the interaction of piperine molecule with DNA duplex. We have carried out UV-vis absorption and fluorescence spectroscopy to confirm the binding of piperine with calf thymus DNA (ctDNA). The energetics of interaction of piperine with ctDNA was monitored by isothermal titration calorimetry (ITC). Differential scanning calorimetry (DSC) and melting temperature (Tm) analysis were also performed, confirming a minor groove mode of binding of piperine with ctDNA. The binding free energy ΔG values obtained from molecular dynamics simulation studies agree well with ITC values and reveal a sequence dependent minor groove binding exhibiting a specificity toward AT rich sequences.

Molecular dynamics simulations of xDNA.

xDNA is a modified DNA, which contains natural as well as expanded bases. Expanded bases are generated by the addition of a benzene spacer to the natural bases. A set of AMBER force-field parameters were derived for the expanded bases and the structural dynamics of the xDNA decamer (xT5' G xT A xC xG C xA xG T3').(xA5' C T xG C G xT A xC A3') was explored using a 22 ns molecular dynamics simulation in explicit solvent. During the simulation, the duplex retained its Watson-Crick base-pairing and double helical structure, with deviations from the starting B-form geometry towards A-form; the deviations are mainly in the backbone torsion angles and in the helical parameters. The sugar pucker of the residues were distributed among a variety of modes; C2' endo, C1' exo, O4' endo, C4' exo, C2' exo, and C3' endo. The enhanced stacking interactions on account of the modification in the bases could help to retain the duplex nature of the helix with minor deviations from the ideal geometry. In our simulation, the xDNA showed a reduced minor groove width and an enlarged major groove width in comparison with the NMR structure. Both the grooves are larger than that of standard B-DNA, but major groove width is larger than that of A-DNA with almost equal minor groove width. The enlarged groove widths and the possibility of additional hydration in the grooves makes xDNA a potential molecule for various applications.

2-[2-Benzoyl-3,3-bis-(methyl-sulfan-yl)prop-2-enyl-idene]malononitrile.

The title compound, C(15)H(12)N(2)OS(2), is an example of a push-pull butadiene in which the electron-releasing methyl-sulfanyl groups and electron-withdrawing nitrile groups on either end of the butadiene chain enhance the conjugation in the system. Short intra-molecular C-H⋯S inter-actions are observed. In the crystal structure, an O⋯C short contact of 2.917 (3) Šis observed.

2-[2-(4-Methyl-benzo-yl)-3,3-bis-(methyl-sulfan-yl)prop-2-enyl-idene]malononitrile.

The title compound, C(16)H(14)N(2)OS(2), is an example of a push-pull butadiene in which the electron-releasing and electron-withdrawing attachments on either end of the butadiene chain enhance the conjugation in the system. The mol-ecules are linked by inter-molecular C-H⋯N inter-actions.

Highly facile and stereoselective intramolecular [2 +2]photocycloadditions of bis(alkenoyl)ketenedithioacetals.

The conformational change induced by the introduction of a ketenedithioacetal moiety at C-4 of 1,7-substituted-1,6-heptadiene-3,5-diones results in favorable spatial relationships between the alkenoyl groups to effect efficient intramolecular cycloadditions: irradiation of bis(alkenoyl)ketenedithioacetals in solution leads to facile and stereospecific intramolecular [2 + 2] photocycloadditions resulting in the formation of substituted bicyclo[3.2.0]heptane-2,4-diones, the observed conformational rigidity of which is attributed to the push-pull character of the ketenedithioacetal group.

Crystal structures of two forms of a 14-mer RNA/DNA chimer duplex with double UU bulges: a novel intramolecular U*(A x U) base triple.

The RNA/DNA 14-mer, (gguauuucgguaCc)2 with consecutive uridine bulges (underlined) on each strand has been determined in two crystal forms, spermine bound (Sp-form) and spermine free (Sp-free). The former was solved by the MAD method with three-wavelength data collected at Brookhaven National Laboratory (BNL); the later isomorphous structure was solved by the molecular replacement method using data collected on our Raxis IIc imaging plate system. The two crystal forms belong to the space group C2 with one molecule of double-stranded 14 mer in the asymmetric unit. The Sp-form has cell constants, a = 60.06, b = 29.10, c = 52.57 A, beta = 120.79 degrees and was refined to 1.7 A resolution with a final Rwork/Rfree of 19.8%/22.7% using 8,549 independent reflections. The Sp-free structure has cell constants, a = 60.06, b = 29.58, c = 52.50 A, beta = 120.85 degrees and was refined to 1.8 A with a final Rwork/ Rfree of 20.8%/23.2% using 6,285 unique reflections. The two structures are identical, except that the Sp-form has a spermine bound in the major groove, parallel to the RNA helical axis. One of the uridine bulges forms a novel intramolecular U*(A x U) base triple. The helices are in the C3'-endo conformation (A-form), but the bulges adopt the C2'-endo sugar pucker. Furthermore, the bulges induce a kink (30 degrees) in the helix axis and a very large twist (55 degrees) between the base pairs flanking the bulges. The Sp-form has one Mg2+ ion whereas the Sp-free form has two Mg2+ ions.

Crystal structure of an RNA duplex r(gugucgcac)(2) with uridine bulges.

The crystal structure of a nonamer RNA duplex with a uridine bulge in each strand, r(gugucgcac)(2), was determined at 1.4 A resolution. The structure was solved by multiple anomalous diffraction phasing method using a three-wavelength data set collected at the Advanced Protein Source and refined to a final R(work)/R(free) of 21.2 %/23.4 % with 33,271 independent reflections (Friedel pairs unmerged). The RNA duplex crystallized in the tetragonal space group P4(1)22 with two independent molecules in the asymmetric unit. The unit cell dimensions are a=b=47.18 A and c=80.04 A. The helical region of the nonamer adopts the A-form conformation. The uridine bulges assume similar conformations, with uracils flipping out and protruding into the minor groove. The presence of the bulge induces very large twist angles (approximately +50 degrees) between the base-pairs flanking the bulges while causing profound kinks in the helix axis at the bulges. This severe twist and the large kink in turn produces a very narrow major groove at the middle of the molecule. The ribose sugars of the guanosines before the bulges adopt the C2'-endo conformation while the rest, including the bulges, are in the C3'-endo conformation. The intrastrand phosphate-phosphate (P-P) distance of the phosphate groups flanking the bulges (approximately 4.4 A) are significantly shorter than the average P-P distance in the duplex (6.0 A). This short distance between the two phosphate groups brings the non-bridging oxygen atoms close to each other where a calcium ion is bound to each strand. The calcium ions in molecule 1 are well defined while the calcium ions in molecule 2 are disordered.

Crystal structure of an adenine bulge in the RNA chain of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag).

Crystal structure of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag), with an adenine bulge in the polypurine RNA strand was determined at 2.3 A resolution. The structure was solved by the molecular replacement method and refined to a final R-factor of 19.9% (Rfree 22.2%). The hybrid duplex crystallized in the space group I222 with unit cell dimensions, a = 46.66 A, b = 47.61 A and c = 54.05 A, and adopts the A-form conformation. All RNA and DNA sugars are in the C3'-endo conformation, the glycosyl angles in anti conformation and the majority of the C4'-C5' torsion angles in g+ except two trans angles, in conformity with the C3'-endo rigid nucleotide hypothesis. The adenine bulge is looped out and it is also in the anti C3'-endo conformation. The bulge is involved in a base-triple (C.g)*a interaction with the end base-pair (C9.g10) in the minor groove of a symmetry-related molecule. The 2' hydroxyl group of g15 is hydrogen bonded to O2P and O5' of g17, skipping the bulged adenine a16 and stabilizing the sugar-phosphate backbone of the hybrid. The hydrogen bonding and the backbone conformation at the bulged adenine site is very similar to that found in the crystal structure of a protein-RNA complex.

An X-ray analysis of native monoclinic lysozyme. A case study on the reliability of refined protein structures and a comparison with the low-humidity form in relation to mobility and enzyme action.

The atomic models of native monoclinic lysozyme obtained by refinement at Bangalore and elsewhere [Young, Dewan, Nave & Tilton (1993). J. Appl. Cryst. 26, 309-319] differed significantly in the flexible regions of the protein molecule. The two models were reconciled starting from regions where they were in reasonable agreement to produce an improved model which yielded an R value of 0.169 for 12 816 observed reflections in the 10-2 A resolution range. The reconciled model was compared with the structure of the 88% relative humidity form obtained through a water-mediated transformation [Madhusudan, Kodandapani & Vijayan (1993). Acta Cryst. D49, 234-245]. Parts of the flexible regions of the molecule register significant movements during the transformation. The changes resulting from the transformation from the native to the low-humidity forms are pronounced in many of the side chains in the active-site region, thus indicating the relationship between hydration, mobility and enzyme action. The fact that the overall changes in molecular geometry resulting from water-mediated transformation are similar to those which occur during enzyme action, further emphasizes this relationship.

Water-dependent domain motion and flexibility in ribonuclease A and the invariant features in its hydration shell. An X-ray study of two low-humidity crystal forms of the enzyme.

The crystal structures of 88 and 79% relative humidity forms of ribonuclease A, resulting from water-mediated transformations, have been refined employing the restrained least-squares method using X-ray data collected on an area detector to R = 0.173 for 15 326 observed reflections in the 10-1.5 A resolution shell and R = 0.176 for 8534 observed reflections in the 10-1.8 A shell, respectively. The comparison of these structures with those of the native, the phosphate-bound and the sulfate-bound forms demonstrates that the mobility of the ribonuclease A molecule involves hinge-bending movement of the two domains and local flexibility within them, particularly at the termini of regular secondary structures and in loops. The comparison also leads to the identification of 31 invariant water molecules in the hydration shell of the enzyme, many of which are involved in holding different parts of the molecule together and in stabilizing local structure. The conformational changes that accompany the partial removal of the surrounding water, particularly those observed in the 79% form, could be similar to those that occur during enzyme action.

Characterization of lysozyme crystals with unusually low solvent content.

Studies on the low-humidity (88%) forms of tetragonal and monoclinic lysozyme, resulting from water-mediated transformations, have provided a wealth of information on the variability in protein hydration, its structural consequences and the water structure associated with proteins, in addition to facilitating the delineation of the rigid and the flexible regions in the protein molecule and the invariant features in its hydration shell. Surprisingly, monoclinic lysozyme continues to diffract even when the environmental humidity is drastically reduced, thus permitting the structural study of the enzyme at different levels of hydration. As part of a study in this direction, three very low humidity forms, two of them occurring at a nominal relative humidity of 38% and the other at 5% relative humidity, have been characterized. These have unprecedented low solvent contents of 16.9, 17.6 and 9.4%, respectively, as determined by the Matthews method.

Crystal structure and conformation of two N-tosyl-protected dipeptides containing amino acids with polar side-chains.

X-Ray diffraction studies and energy-minimization calculations were carried out on two dipeptides, N-tosyl-L-Ser-Gly-OMe monohydrate (C13H18N2O6S.H2O, compound A) and N-tosyl-L-Thr-Gly-OMe (C14H20N2O6, compound B). Compound A crystallized in the monoclinic system, space group P2(1) with unit cell parameters a = 4.915(1), b = 15.625(4), c = 11.003(1) A, beta = 91.28(1) degrees, V = 844.8 A3. M(r) = 348.4, d = 1.37(2) g cm-3, Z = 2, lambda(Cu K alpha) = 1.5418 A, mu = 1.99 mm-1, T = 293 K. R = 0.032 for 1451 unique reflections with I > 2 sigma(I). Compound B crystallized in the orthorhombic system, space group P2(1)2(1)2(1), with unit cell parameters a = 5.050(2), b = 16.483(3), c = 20.769(5) A, V = 1729.3 A3, Z = 4. M(r) = 344.4, d = 1.32(2) g cm-3, mu(Cu K alpha) = 1.90 mm-1. R = 0.040 for 1060 unique reflections with I > 2 sigma(I). The major difference in the backbone conformation of the two compounds is in their glycine residues, with the glycine residue in compound A adopting an extended conformation with phi = -132.6(3) degrees and psi = 175.3(3) degrees and that in compound B having a folded conformation with phi = -56.3(6) degrees and psi = -42.6(7) degrees. In compound A the oxygen atom of the Ser side-chain and the carbonyl oxygen atom of glycine are bridged by the water of crystallization through O--H ... O hydrogen bonds, resulting in the relatively rare trans conformation [chi = -175.7(2) degrees] for this side-chain.(ABSTRACT TRUNCATED AT 250 WORDS)

C9 conformation of N-(N alpha-[(tert.-butyloxy)-carbonyl]-L-alanyl)-N,N'-dicyclohexylu rea in solid and solution.

An X-ray diffraction study was carried out on a single crystal of N-(N alpha-[(tert.-butyloxy)-carbonyl]-L-alanyl)-N,N'-dicyclohexylur ea belonging to the tetragonal space group P4(1)2(1)2, having cell dimensions a = b = 10.102(3) A, c = 46.067(7) A, V = 4701.2 A3, Z = 8. The crystal structure was solved by direct methods and refined to an R value of 0.056 for 1602 unique reflections with I greater than 2.5 sigma(I). Crystal structure analysis shows the presence of an intramolecular N-H ... O=C H-bond stabilizing the molecule in a folded form similar to that of a beta turn, forming a nine-membered ring. IR and 1H-NMR studies in CDCl3 solution confirm the stable folded conformation found in the crystalline state, as well as the existence of N-H ... O=C H-bonds in the title compound, as in peptides.