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Large unilamellar lipid vesicles do not normally fuse under fluid shear stress. They might deform and open pores to relax the tension to which they are exposed, but membrane fusion occurring solely due to shear stress has not yet been reported. We present evidence that shear forces in a viscous solution can induce lipid bilayer fusion. The fusion of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes is observed in Couette flow with shear rates above 3000 s(-1) provided that Couette Cell the medium is viscous enough. Liposome samples, prepared at different viscosities using a 0-50 wt % range of sucrose concentration, were studied by dynamic light scattering, lipid fusion assays using Forster resonance energy transfer (FRET), and linear dichroism (LD) spectroscopy. Liposomes in solutions with 40 wt % (or more) sucrose showed lipid fusion under shear forces. These results support the hypothesis that under suitable conditions lipid membranes may fuse in response to mechanical-force-induced stress.

We report here how the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, simultaneously measuring changes in the induced energy dissipation, D (cf. viscoelastic properties), and the frequency, f (cf. coupled mass), can be used to characterize the bound state of single-stranded peptide nucleic acid (PNA) and deoxyribose nucleic acid (DNA) in relation to their ability to function as selective probe(s) for fully complementary and single-mismatch DNA. The possibility to use the QCM-D technique for detection of binding kinetics and structural differences in the formed duplexes is also presented. We found that thiol-PNA and thiol-DNA attached via a sulfur group directly on a bare-gold surface are less efficient as probes for DNA than are biotin-PNA and biotin-DNA, coupled on top of a two-dimensional (2-D) arrangement of streptavidin, formed on a biotinylated phospholipid bilayer on a SiO2 surface. The fully complementary and singly mismatched DNA oligomers hybridize with the immobilized PNA and DNA. A single mismatch is discriminated via a significant difference in the binding and dissociation kinetics, demonstrating a high selectivity and thus successful immobilization of functional single strands. The observed ratios between hybridization-induced energy dissipation (DeltaD) and the frequency shift (Deltaf) made it possible to discriminate thiol-PNA directly attached to a gold surface from biotin-PNA coupled to the streptavidin 2-D arrangement, where the former were shown to be inefficient for detecting subsequent hybridization. Structural differences of the immobilized layers composed of biotin-PNA-DNA and biotin-DNA-DNA were clearly reflected by the DeltaD and Deltaf response.

Sequence-specific binding to genomic-size DNA sequences by artificial agents is of major interest for the development of gene-targeting strategies, gene-diagnostic applications, and biotechnical tools. The binding of one such agent, peptide nucleic acid (PNA), to a randomized human genome has been modeled with statistical mass action calculations. With the length of the PNA probe, the average per-base binding constant k(0), and the binding affinity loss of a mismatched base pair as main parameters, the specificity was gauged as a "therapeutic ratio" G = maximum safe [PNA](tot)/minimal efficient [PNA](tot). This general, though simple, model suggests that, above a certain threshold length of the PNA, the microscopic binding constant k(0) is the primary determinant for optimal discrimination, and that only a narrow range of rather low k(0) values gives a high therapeutic ratio G. For diagnostic purposes, the value of k(0) could readily be modulated by changing the temperature, due to the substantial Delta H degrees associated with the binding equilibrium. Applied to gene therapy, our results stress the need for appropriate control of the binding constant and added amount of the gene-targeting agent, to meet the varying conditions (ionic strength, presence of competing DNA-binding molecules) found in the cell.

A series of ruthenium(II) complexes comprising differently substituted dipyridophenazine (dppz) ligands have been examined with respect to their orientation when bound to phosphatidylcholine liposome bilayers. The liposomes were subjected to shear flow, thereby deformed into ellipsoid shapes, and the orientation of chromophores in the bilayers could then be examined with polarized light (linear dichroism, LD). After correction for turbidity dichroism, LD distinguishes between chromophore transition moments oriented along the lipid chains (negative LD), and parallel to the membrane surface (positive LD). The polarizations, energies, and absorption overlap of the electronic transitions of the ruthenium complexes, known from previous work, could be used to characterize the orientation of liposome bound complexes. From luminescence lifetime studies further information about the localization of the chromophores was obtained. Assuming that the positively charged metal moiety of the complexes remains close to th...

A library of thrombin inhibitors has been designed using statistical molecular design. An aromatic scaffold was used, with three varied positions corresponding to three pockets at the active site of thrombin (the S-, P-, and D-pockets). The selection was performed in the building block space, and previously acquired data were included in the design procedure. The design resulted in six, four, and six building blocks for the first (S), second (P), and third (D) pockets, respectively. A second round of selection applied to the combined selected building blocks resulted in a subset of 18 compounds. The selected library was synthesized in parallel and biologically evaluated. The compounds were analyzed with respect to their inhibition (pIC(50)) of thrombin; membrane permeability, estimated by migration behavior in micellar media (CE log k') and pK(a); and specificity with respect to inhibition (K(i)) of trypsin. Multivariate QSAR studies of the responses yielded valuable results and information that could only be found using statistical molecular design in combination with multivariate analysis.

The interaction of the cellular delivery vector penetratin with a model system consisting of negatively charged phospholipid vesicles has been studied. Above a certain peptide to lipid molar ratio, the cationic oligopeptide induces vesicle aggregation. Interestingly, the aggregation is followed by spontaneous disaggregation, which may be related to membrane translocation of the peptide. Circular dichroism (CD) measurements indicate a conformational transition, from alpha -helix to antiparallel beta -pleated sheet, which is simultaneous with the aggregation process. The potential influence of spectroscopic artifacts on CD data due to the drastically increased turbidity during aggregation is discussed.

The source of the activation energy that allows cutting of DNA by restriction enzymes is unclear. A systematic study of the cutting efficiency of the type-II restriction endonuclease EcoRI, with varying background electrolyte ion pair and buffer reported here, shows only a modest dependence of efficiency on cation type. Surprisingly, efficiency does depend strongly on the presumed indifferent anion of the background salt. What emerges is that competition between the background salt anion and the buffer anion for the enzyme and DNA surfaces is crucial. The results are unexpected and counterintuitive from the point of view of conventional electrolyte theory. However, taken together with recent developments in surface chemistry, the results do fall into place and could also suggest a novel mechanism for enzyme activity as an alternative to metal-activated hydrolysis: microscopic cavitation in a hydrophobic pocket might be the source of activation energy.

The interactions between the stereoisomers of the chiral bis-intercalator [mu -C4(cpdppz)(2)-(phen)(4)Ru-2](4+) and DNA reveal interesting dynamic discrimination properties. The two enantiomers Delta-Delta and Lambda-Lambda both form very strong complexes with calf thymus DNA with similar thermodynamic affinities. By contrast, they display considerable variations in their binding kinetics. The Delta-Delta enantiomer has higher affinity for calf thymus DNA than for [poly(dA-dT)](2), and the association kinetics of the dimer to DNA, as well as to polynucleotides, requires a multiexponential fitting function. The dissociation reaction, on the other hand, could be described by a single exponential for [poly(dA-dT)](2), whereas two exponentials were required for mixed-sequence DNA. To understand the key mechanistic steps of the reaction, the kinetics was studied at varied salt concentration for different choices of DNA and chirality of the threading complex. The enantiomers were found to have markedly different dissociation rates, the Lambda-Lambda enantiomer dissociating about an order of magnitude faster than the Delta-Delta enantiomer. Also. the salt dependence of the dissociation rate constants differed between the enantiomers, being stronger for the Lambda-Lambda enantiomer than for the Delta-Delta enantiomer. Since the dissociation reaction requires unthreading of bulky parts of the bis-intercalator through the DNA helix, a considerable conformational change of the DNA must be involved, possibly defining the rate-limiting step.

Currently there is an extensive search for biosensors for detecting genetic defects by hybridisation to immobilised oligonucleotides. A concept able to detect a single mismatch in a 15mer single-strand target of the p53 tumor suppresser gene is presented (a mutation found in many types of cancer cells). In this method, the unique hybridisation properties of the DNA mimic peptide nucleic acid (PNA) are combined with electronically detected mass and shear dissipation at the surface of a quartz crystal. Cysteine-labeled PNA efficiently immobilises on crystal-gold surface at 20 degrees C. At 60 degrees C, addition of complementary DNA results in a signal response corresponding to hybridisation between DNA and the PNA-covered surface, whereas addition of DNA differing in only one of the 15 bases gives no response at all.

Two novel transmembrane fluorescent diester probes D and E bearing an anthracenediyl moiety in the middle of the molecule have been synthesized. Their absorption and fluorescence spectra in CHCl3 solution as well as their fluorescence characteristics in dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles were determined. Although their absorption spectra (first transition, S-0 --> S-1) present a good overlap with the fluorescence spectrum of tryptophan, only probe E could be a good acceptor for the energy-transfer experiments, since a strong overlap exists between the absorption spectrum of tryptophan and the second transition (S-0 --> S-2) of the absorption spectrum of probe D. The Forster critical distance R-0 for energy transfer between tryptophan (donor) and probe E (acceptor) is found to be 23-24 Angstrom. Finally, linear-dichroism studies on shear-deformed DMPC vesicles show the incorporated probe E to lie essentially perpendicular to the bilayer plane. These results establish that probe E could be useful in the study of membrane-bound protein topography by the fluorescence-energy-transfer method.

In this paper we report the DNA binding proper ties of piperazinecarbonyloxy-2-propyl derivatives of anthracene (2), pyrene (3), and phenylanthracene (4). An intercalative binding mode is found for 2 and 3, while the phenyl group of 4 prevents intercalation and leads to external binding. Preferential binding of the (S)-enantiomers is found for both anthracene 2 and pyrene 3. However, the enantiomeric preference is small, with K-(R)/K-(S) being around 0.5 for both the anthracene and the pyrene compounds. This is interpreted in terms of orientation polarity in the binding, by which any intrinsic enantioselectivity is canceled by averaging of opposite binding orientations. The affinities for poly(dA-dT)(2) (AT) are 10(4) M-1 for anthracene derivative 2, and 5 x 10(5) M-1 for pyrene derivative 2. The affinities for poly(dG-dC)(2) (GC) are I order of magnitude lower than those for AT. This is explained by steric interference of the piperazinium tail with the exocyclic amino groups of guanine in the minor groove of GC, leading to a more shallow intercalation in GC than in AT, as also indicated by significantly less negative reduced linear dichroism of the intercalator absorption bands in the GC complexes. This behavior is consistent with that observed for the previously studied achiral analogues.(1) Binding thermodynamics support the difference in binding mode between AT and GC. The binding enthalpy of the AT complexes is significantly more negative than that of the corresponding GC complexes. This indicates a larger overlap of intercalating moiety and nucleobases in the AT complexes, consistent with the linear dichroism results.

Synthetic molecules that can bind with high sequence specificity to a chosen target in a gene sequence are of major interest in medicinal and biotechnological contexts. They show promise for the development of gene therapeutic agents, diagnostic devices for genetic analysis, and as molecular tools for nucleic acid manipulations. Peptide nucleic acid (PNA) is a nucleic acid analog in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone usually formed from N-(2-amino-ethyl)-glycine units, resulting in an achiral and uncharged mimic. It is chemically stable and resistant to hydrolytic (enzymatic) cleavage and thus not expected to be degraded inside a living cell. PNA is capable of sequence-specific recognition of DNA and RNA obeying the Watson-Crick hydrogen bonding scheme, and the hybrid complexes exhibit extraordinary thermal stability and unique ionic strength effects. It may also recognize duplex homopurine sequences of DNA to which it binds by strand invasion, forming a stable PNA-DNA-PNA triplex with a looped-out DNA strand. Since its discovery, PNA has attracted major attention at the interface of chemistry and biology because of its interesting chemical, physical, and biological properties and its potential to act as an active component for diagnostic as well as pharmaceutical applications. In vitro studies indicate that PNA could inhibit both transcription and translation of genes to which it has been targeted, which holds promise for its use for antigene and antisense therapy. However, as with other high molecular mass drugs, the delivery of PNA, involving passage through the cell membrane, appears to be a general problem.

A combination of measurement of mobility and orientational dynamics of long reptating DNA in agarose gel has been used to reveal how the binding of Delta and Lambda enantiomers of the tris(phenanthroline)ruthenium(II) ion affects the flexibility of the DNA helix. The mobility data, and data over the step length and period time of the reptation cycle, obtained in the presence of the respective enantiomer, are compared with those of free DNA and with data obtained earlier on DNAs with known variations in helix flexibility. The results suggest that the Delta form induces kinks in the DNA helix while the Lambda form gives rise to a local stiffening of the helix.

Despite exhaustive chemical and crystal structure studies, the mechanistic details of how F(o)F(1)-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent high-resolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO(3)(-) ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the gamma-subunit rotation, may effectively block the back reaction in beta(TP) and, thus, promote ATP.

Two novel substitutionally-inert diastereomeric ruthenium(II) cations of the form Lambda- and Delta-cis-beta-[Ru(RR-picchxn)(phen)](2+), where RR-picchxn is N,N'-dimethyl-N,N'-di(2-picolyl)-IR,2R-diaminocyclohexane and phen is 1,10-phenanthroline, have been studied with respect to their interactions with duplex DNA. NMR investigations show that both diastereomers bind to the oligonucleotide [d(CGCGATCGCG)](2) in the fast exchange regime and that binding predominantly takes place in the minor groove of the oligonucleotide, but that the governing interactions are significantly different for the two Delta and Lambda forms. Linear dichroism data support the latter interpretation, in that the relative orientations of cis-beta-[Ru(RR-picchxn)(phen)](2+) to calf thymus DNA also are observed to differ for the Delta and Lambda diastereomers. Interpretation of these data indicates the Lambda form to be bound with the planar phen ligand closely parallel to the DNA base-pairs, but the average orientation of the phen ligand in the Delta form deviates significantly from a parallel alignment.

Four novel piperazinylcarbonyloxyethyl derivatives of anthracene and pyrene have been prepared and investigated with respect to sequence specificity and synergism between hydrophobic and electrostatic effects upon binding to DNA. Linear and circular dichroism spectroscopy was used to assess the orientation of the aromatic chromophores relative to the nucleobases. Anthracene and pyrene derivatives 2a and 3 are both concluded to bind to homo-polynucleotide poly(dA-dT)(2) by intercalation of their aromatic moieties between base pairs, with a binding constant K-AT of 4 x 10(5) M-1 and 2 x 10(6) M-1, respectively. Significantly reduced affinities (K-GC = 3 x 10(4) M-1 and 10(5) M-1, respectively) are observed with poly(dG-dC)(2), due to less favorable interactions of the piperazinium tail in the minor groove. Base pair specificity is reflected in the binding thermodynamics, with the binding to AT being more enthalpically driven than the binding to GC. Phenyl substitution at the quaternary piperazinium site of the anthracene derivative 2b, does not affect the ratio K-AT/K-GC, but reduces the affinity for both AT End GC slightly. Moreover, the phenyl group in the 10-position of 4 prevents intercalation, and apparently, this compound binds externally to both AT and GC duplex polynucleotides. The results are discussed in terms of general features of the interactions of the intercalating and minor-groove binding molecular moieties, and their interplay with each other, with potentials for tuning specificity.

The binding conformations of single anti- and syn-BPDE-N2-dG adducts in oligonucleotides of varying base composition have been studied by induced circular dichroism (ICD) and light absorption spectroscopy. The sign of the ICD in single-stranded oligonucleotide adducts correlates with the absolute configuration of the cyclohexyl moiety of the BPDE. Adducts in oligonucleotide duplexes with UV lambdamax350 nm exhibiting a significant duplex-induced positive ICD should have a minor groove location as the predominant conformation. Those with UV lambdamax350 nm exhibiting either positive or negative contributions to the ICD should have intercalated binding as the predominant conformation. The magnitude of the ICD is dependent on the sequence context of the adducted strand and the particular BPDE-adduct isomer under study. In some cases, the results suggest structural heterogeneity. For instance, the (+)- and the (-)-trans-anti-BPDE-N2-dG adducts in duplexes where a dT flanks the lesion site exhibit weak positive ICD or negative ICD. These results reflect a bimodal conformational adduct distribution with contributions from both externally and internally located adducts. A key observation for the (+)-cis-syn-BPDE-N2-dG complexes in 5'-d(TGC) and 5'-d(CGC) sequence contexts is that the near-UV absorption spectra showed distinct bands corresponding to minor groove binding (lambdamax congruent with 346 nm) as well as intercalative binding (lambdamax congruent with 354 nm). Evidence for an equilibrium between the different modes of localization is provided by the results from the temperature dependence of the near-UV absorption and ICD characteristics of (+)-cis-syn-BPDE-N2-dG complexes in 5'-d(TGC) and 5'-d(CGC) sequence contexts, respectively.

The L2 loop in the RecA protein is the catalytic center for DNA strand exchange. Here we investigate the DNA binding properties of the L2 loop peptide using optical spectroscopy with polarized light. Both fluorescence intensity and anisotropy of an etheno-modified poly(dA) increase upon peptide binding, indicate that the base motions of single-stranded DNA are restricted in the complex. In agreement with this conclusion, the peptide-poly(dT) complex exhibits a significant linear dichroism signal. The peptide is also found to modify the structure of double-stranded DNA, but does not denature it. It is inferred that strand separation may not be required for the formation of a joint molecule.

The electronically excited states of the phthalimide chromophore have been studied by means of linear dichroism (LD) of samples partially oriented in poly(vinyl alcohol) films, magnetic circular dichroism (MCD), and circular dichroism (CD) spectroscopy. On the basis of the LD measurements, the low-energy tail (340-320 nm) of the first absorption band is assigned to an out-of-plane polarized pi-->pi* transition (I). At higher energy, the electronic spectrum is resolved into contributions from five pi-->pi* transitions: II(300 nm, long-axis polarized), III (275 nm, short-axis polarized), IV (235 nm, short-axis polarized), V (220 nm, long-axis polarized), and VI (similar to 210 nm, short-axis polarized). The results from semiempirical (INDO/S-CI) and ab initio (CIS/6-31+G(d)) MO calculations compare well with the proposed assignments of the excited states. Degenerate exciton interaction between electric-dipole-allowed transitions of two phthalimide chromophores is observed in the electronic absorption spectra of the achiral bis-phthalimides 2a-c and in the CD spectrum of the chiral bis-phthalimide 3a. For the latter compound, the solid-state geometry has been determined by X-ray diffraction analysis. Good agreement between experimental and computed CD spectra confirms that the coupled-oscillator exciton model provides the basis for a reliable nonempirical method for the assignment of absolute configuration for this class of compounds. Nondegenerate exciton coupling between phthalimide and benzoate or phenyl chromophores is born out in the CD spectra of homochiral molecules 3c and 3d with the rigid cyclohexane skeleton. Finally, the exciton coupling method is used to make stereochemical assignments for the acyclic, conformationally flexible derivatives 4a-c and 5b.

Norfloxacin, a quinolone antibacterial reagent, has been studied with respect to its binding to calf thymus DNA using fluorescence and linear dichroism techniques and unwinding of supercoiled DNA. The fluorescence of norfloxacin is strongly quenched in the presence of DNA and using this decrease in a fluorescence titration the equilibrium constant of the complex formation was established to be 2.8 x 10(3) M-1. The electric transition moments of the norfloxacin chromophore have been analyzed using fluorescence anisotropy, magnetic circular dichroism, and linear dichroism in stretched poly(vinyl alcohol) film and INDO/S calculations. These data are then used to interpret flow linear dichroism results for the norfloxacin-DNA complex. The transition moments for the long-wavelength transitions are found to be oriented at about 65.0-85.0 degrees with respect to the DNA helix axis. A near perpendicular orientation of the norfloxacin chromophore plane makes it possible to exclude classical groove or surface binding modes. The possibility of a classical intercalation binding mode also can be ruled out from unwinding experiments. However, it is shown that the molecular plane of norfloxacin is near perpendicular relative to the DNA helix axis with a possibility of a bending of the DNA helix at the binding site.

To gain insight into the mechanism of pairing two complementary DNA strands by the RecA protein, we have determined the nucleobase orientation of the first and the second bound DNA strands in the RecA DNA filament by combined measurements of linear dichroism and small angle neutron scattering on flow-oriented samples. An etheno-modified DNA, poly(d epsilon A) was adapted as the first DNA and an oligo(dT) as the second DNA, making it possible to distinguish between the linear dichroism signals of the two DNA strands, The results indicate that binding of the second DNA does not alter the nucleobase orientation of the first bound strand and that the bases of the second DNA are almost coplanar to the bases of the first strand although somewhat more tilted (60 degrees relative to the fiber axis compared with 70 degrees for the first DNA strand). Similar results were obtained for the RecA DNA complex formed with unmodified poly(dA) and oligo(dT). An almost coplanar orientation of nucleobases of two DNA strands in a RecA-DNA filament would facilitate scanning for, and recognition of, complementary base sequences. The slight deviation from co-planarity could increase the free energy of the duplex to facilitate dissociation in case of mismatching base sequences.

It has recently been found that 2,7-diazapyrenes upon interaction with nucleic acids form stacked (“intercalation”) complexes, which for the methylated derivatives exhibit new absorption features assigned as charge-transfer (CT) transitions.1 To better understand the basis of these interactions and associated optical properties, the geometries and electronic spectra of complexes of adenine (A) with 2,7-diazapyrene (DAP), N-methyl-2,7-diazapyrenium (MDAP+), and N,N‘-dimethyl-2,7-diazapyrenium (DMDAP2+) have been modeled using semiempirical AM1 and PM3 geometry optimizations, ab initio (vacuum and Onsager model) energy calculations, and ZINDO/S calculations. In addition, absorption spectra, fluorescence quenching, and 1H NMR spectra for the complexes in aqueous solution have been measured. For the A-DAP complex, a coplanar, hydrogen-bonded complex is predicted by the calculations, while A-MDAP+ and A-DMDAP2+ complexes should have edge-to-face geometry. The association is predicted to be of electrostatic nat...

The binding of 2,7-diazapyrene (DAP), N-methyl-2,7-diazapyrenium monocation (MDAP), and N,N‘-dimethyl-2,7-diazapyrenium dication (DMDAP) to calf thymus DNA has been studied with respect to molecular geometry and thermodynamics. It is concluded from flow linear dichroism (LD) and induced circular dichroism (CD) spectra that the three diazapyrenes bind by intercalation to alternating AT as well as GC polynucleotide duplexes, as indicated by strong interactions with the transitions of the nucleobases in conjunction with approximately perpendicular orientations of the in-plane symmetry axes relative to the DNA helix axis. The reduced LD (LDr = LD/Aiso) of the DNA complexes is characterized by marked fine structure, decreasing in the order DAP > MDAP > DMDAP. This finding is interpreted in terms of a microscopic heterogeneity associated with rotational mobility of the ligand in a tilted intercalation pocket, with the dication DMDAP having less rotational freedom than the neutral DAP has. Other distinct differe...

The electronic transitions giving rise to the UV-visible absorption spectra of two pyridinium-phenanthridinium viologens, 6,7-dihydropyridol[2',1':3,4]pyrazinol[1,2-f]phenanthridinediium dication (1) and 7,8-dihydro-6H-pyrido[2',1':3,4]diazepino[1,2-f]phenanthridinediium dication (2), have been investigated with respect to energies, intensities, and transition moment directions. A combination of methods has been applied: UV-visible absorption, circular dichroism, magnetic circular dichroism, fluorescence anisotropy, Linear dichroism In stretched poly(vinyl alcohol) films, and semiempirical molecular orbital calculations. For both drugs, the lowest energy absorption band, occurring around 400 nm, results from two separate transitions. The corresponding electric transition dipole moments lie in the phenanthridine plane and are polarized, respectively, in the direction of the pyridine moiety (the lower energy transition) and parallel to the phenanthridine long axis (the higher energy transition). Up to four additional different pi --> pi* transitions account for a second band that peaks at 250 nm; they show different polarizations within the phenanthridine plane. The lowest energy transition of the whole spectrum of both drugs corresponds to the promotion of an electron from the HOMO to the LUMO, which are molecular orbitals mainly localized in the phenanthridine and pyridine rings, respectively, thereby implying a charge transfer, upon excitation, from the phenanthridine toward the pyridine ring. The experimental and theoretical results are discussed in relation to the spectroscopic, redox, and photochemical properties of these drugs.

The binding of a mixed-sequence pentadecamer PNA (peptide nucleic acid) containing all four nucleobases to the fully complementary as well as various singly mismatched RNA and DNA oligonucleotides has been systematically investigated using thermal denaturation and BIAcore surface-interaction techniques. The rate constants for association (k(a)) and dissociation (k(d)) of the duplex formation as well as the thermal stability (melting temperature, T(m)) of the duplexes have been determined. Upon binding to PNA tethered via a biotin-linker to streptavidin at the dextran/gold surface, DNA and RNA sequences containing single mismatches at various positions in the center resulted in increased dissociation and decreased association rate constants. T(m) values for PNA x RNA duplexes are on average 4 degrees C higher than for PNA x DNA duplexes and follow quantitatively the same variation with mismatches as do the PNA x DNA duplexes. Also a faster k(a) and a slower k(d) are found for PNA x RNA duplexes compared to the PNA x DNA duplexes. An overall fair correlation between T(m), k(a), and k(d) is found for a series of PNA x DNA and PNA x RNA duplexes although the determination of k(a) seemed to be prone to artifacts of the method and was not considered capable of providing absolute values representing the association rate constant in bulk solution.

The electronic spectrum of the unnatural nucleic acid base 2-aminopurine is important in order to understand the spectroscopy and photophysics of the normal DNA bases. Knowledge of transition moment directions and effective absorption components is also a prerequisite for the interpretation of emission spectra of 2-aminopurine incorporated into DNA as a probe of nucleic acid base motion or excitation energy transfer processes. Using linear dichroism, fluorescence anisotropy, and ordinary and magnetic circular dichroism techniques, the near-UV spectrum of 2-aminopurine is resolved into contributions from five electronic transitions. Four moderately strong pi-->pi* transitions (I, III, IV, and V) are observed, polarized at +53 degrees (I), -72 degrees (III), -77 degrees (IV), and +57 degrees or -67 degrees (V) relative to the short molecular axis. In addition, one weak n --> pi* transition (II) polarized perpendicular to the molecular plane has been identified. Semiempirical INDO/S calculations support these assignments.

The binding of homochiral [Ru(II)(1,10-phenanthroline)(2)L](2+) complexes {where L = 1,10-phenanthroline (phen), dipyrido[3,2-a:2',3'-c]phenazine (DPPZ) or benzodipyrido[3,2-a:2',3'-c]phenazine (BDPPZ)} to poly(dT*dA-dT) triplex has been investigated by linear and circular dichroism and thermal denaturation. Analysis of the linear dichroism spectra indicates that the extended DPPZ and BDPPZ ligands lie approximately parallel to the base-pair and base-tripler planes consistent with intercalation which is also supported by strong hypochromism in the interligand absorption bands with either duplex or tripler. The spectral properties of any of the metal complex enantiomers were similar for binding to either duplex or tripler DNA, indicating that the third strand, which occupies the major groove of the template duplex, has little effect on the binding geometries and hence supports the hypothesis that the metal complexes all bind from the minor groove with the DPPZ and BDPPZ ligands intercalated but without intercalation in the case of [Ru(phen)(3)](2+). Third-strand stabilization depended on the nature of the third substituted phenanthroline chelate ligand but was not directly related to its size, with stabilizing power increasing in the order phen < BDPPZ < DPPZ. This observation further supports intercalation of the extended ligands from the minor groove of the tripler since the extended BDPPZ ligand that would protrude into the major groove of the template would have greater steric interference than DPPZ with the third DNA strand.

Background Oct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. Protein-based reprogramming methods mainly rely on the addition of a fused cell penetrating peptide. This study describes that Oct4 inherently carries a protein transduction domain, which can translocate into human and mouse cells. Results A 16 amino acid peptide representing the third helix of the human Oct4 homeodomain, referred to as Oct4 protein transduction domain (Oct4-PTD), can internalize in mammalian cells upon conjugation to a fluorescence moiety thereby acting as a cell penetrating peptide (CPP). The cellular distribution of Oct4-PTD shows diffuse cytosolic and nuclear staining, whereas penetratin is strictly localized to a punctuate pattern in the cytoplasm. By using a Cre/loxP-based reporter system, we show that this peptide also drives translocation of a functionally active Oct4-PTD-Cre-fusion protein. We further provide evidence for translocation of full length Oct4 into human and mouse cell lines without the addition of any kind of cationic fusion tag. Finally, physico-chemical properties of the novel CPP are characterized, showing that in contrast to penetratin a helical structure of Oct4-PTD is only observed if the FITC label is present on the N-terminus of the peptide. Conclusions Oct4 is a key transcription factor in stem cell research and cellular reprogramming. Since it has been shown that recombinant Oct4 fused to a cationic fusion tag can drive generation of iPSCs, our finding might contribute to further development of protein-based methods to generate iPSCs. Moreover, our data support the idea that transcription factors might be part of an alternative paracrine signalling pathway, where the proteins are transferred to neighbouring cells thereby actively changing the behaviour of the recipient cell. Electronic supplementary material The online version of this article (doi: 10.1186/2045-9769-3-2) contains supplementary material, which is available to authorized users.

In recent years, multi-lipid bicellar systems have emerged as promising membrane models. The fast orientational diffusion and magnetic alignability made these systems very attractive for NMR investigations. However, their alignment was so far achieved with a strong magnetic field, which limited their use with other methods that require macroscopic orientation. Recently, it was shown that bicelles could be aligned also by shear flow in a Couette flow cell, making it applicable to structural and biophysical studies by polarized light spectroscopy. Considering the sensitivity of this lipid system to small variations in composition and physicochemical parameters, efficient use of such a flow-cell method with coupled techniques will critically depend on the detailed understanding of how the lipid systems behave under flow conditions. In the present study we have characterized the flow alignment behavior of the commonly used dimyristoyl phosphatidylcholine/dicaproyl phosphatidylcholine (DMPC/DHPC) bicelle system, for various temperatures, lipid compositions, and lipid concentrations. We conclude that at optimal flow conditions the selected bicellar systems can produce the most efficient flow alignment out of any lipid systems used so far. The highest degree of orientation of DMPC/DHPC samples is noticed in a narrow temperature interval, at a practical temperature around 25 C, most likely in the phase transition region characterized by maximum sample viscosity. The change of macroscopic orientation factor as function of the above conditions is now described in detail. The increase in macroscopic alignment observed for bicelles will most likely allow recording of higher resolution spectra on membrane systems, which provide deeper structural insight and analysis into properties of biomolecules interacting with solution phase lipid membranes. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

The interactions between anionic or zwitterionic liposomes and a water-soluble, DNA-binding photochromic spiropyran are studied using UV/vis absorption and linear dichroism (LD) spectroscopy. The spectral characteristics as well as the kinetics of the thermal isomerization process in the absence and presence of the two different liposome types provide information about the environment and whether or not the spiropyran resides in the liposome membrane. By measuring LD on liposomes deformed and aligned by shear flow, further insight is obtained about interaction and binding geometry of the spiropyran at the lipid membranes. We show that the membrane interactions differ between the two types of liposomes used as well as the isomeric forms of the spiropyran photoswitch.

Linear dichroism (LD) has been used to probe the binding geometries of the diastereomeric adducts between DNA and the Delta and Lambda enantiomers of [Ru(phen)(2)L](2+) where the chelate ligand L is either dipyrido[3,2-a:2',3'-c]-phenazine (DPPZ) or its benzologue benzodipyrido[a:3,2-h:2'3'-j]phenazine (BDPPZ). By combined use of LD and emission anisotropy excitation spectra measured in highly viscous solution the absorption envelope of the metal-to-ligand charge transfer (MLCT) region has been fur the first time resolved in a complex of this type bound to DNA. The absorption can be described by four effective polarization directions (three almost orthogonal), along one of which any transition is polarized, assignments which are in good agreement with molecular orbital (INDO/S) calculations. The analysis of the LD spectrum provides the orientation of the complex in terms of several angles. Near perpendicularity relative to the DNA helix axis, found for the in-plane long and short axes of BDPPZ and DPPZ, together with extensive hypochromicity of the corresponding intraligand transitions, supports intercalation of the (B)DPPZ ligands between the DNA bases as earlier studies have indicated. MLCT transition moments that make an oblique angle to the molecular plane of the (B)DPPZ chromophores confirm this orientation and, in addition, provide strong evidence for a rotation (roll) of the complex around the pseudo-dyad axis, The roll is small (varying between 5 and 15 degrees), but significant, and has the same sign (clockwise) for both enantiomeric forms of the two complexes studied and shows only minor variations between calf-thymus DNA and alternating GC or AT homopolymer duplexes, It may reflect a property intrinsic of DNA (tilt of bases) or be a result of steric interference of the two phenanthroline ''propeller blades'' with a groove. The roll provides the first example of an angle in a DNA system that has been determined with its sign from LD spectroscopy.

Peptide nucleic acid (PNA) is a DNA analogue in which the negatively charged sugar phosphate backbone has been substituted by uncharged N-(2-aminoethyl)glycine units. The study of a PNA-DNA duplex and the corresponding DNA-DNA duplex gives a unique opportunity to compare two polyelectrolytes with virtually identical geometry but greatly different linear charge density. The results provide a basis for a study of the applicability of the Poisson-Boltzmann (PB) and counterion condensation (CC) theories. UV and circular dichroism spectroscopy as well as isothermal titration calorimetry (ITC) have been used to study the effect of different ions on the stability and conformation of PNA-DNA, PNA-PNA, and DNA-DNA duplexes having the same base sequences. Cations in general destabilize both antiparallel (N/3') and parallel (N/5') PNA-DNA duplexes whereas they stabilize the DNA-DNA duplex. Studies on the effect of monovalent salt such as NaCl on T-m were carried out over a wide range of salt concentrations (0.01 to 5 M). The decrease in the T-m of the N/3' PNA-DNA duplex with increasing ionic strength in the range of concentrations of 0.01 to 0.5 M, where electrostatic effects predominate, is explained in terms of counterion release upon duplex formation in contrast to the counterion association accompanying the formation of a DNA duplex. The uncharged PNA-PNA duplex shows no significant destabilization in this concentration range. The higher stability of the N/3' PNA-DNA compared to the DNA-DNA duplex (Delta Delta G similar to-7 kcal/mol) is ascribed to more favorable entropic contributions consistent with the counterion release that accompanies the PNA-DNA duplex formation. At high salt concentration (>1 M), where electrostatic contributions saturate, similar trends in the decrease in T-m, were observed for the three types of duplexes irrespective of their backbone charges. The destabilizing effects of a series of Na salts with various monovalent anions on N/3' PNA-DNA and PNA-PNA duplexes were found to follow the Hofmeister series, emphasizing the importance of the hydrophobic interaction between nucleobases for the stability of the PNA complexes in high salt concentration.

Homopyrimidine PNA can recognize a purine target of double-stranded DNA by local displacement of the DNA pyrimidine strand and formation of a PNA-DNA-PNA tripler. In order to elucidate the mechanism of this PNA/DNA recognition reaction we have studied the interaction of PNA-T oligomers with poly(da):poly(dT) as a model system. The PNA binding kinetics can be observed in situ by spectroscopy as a change in the circular dichroism spectrum. The overall reaction rate decreases with increasing ionic strength and proceeds in minutes at 50 mM NaCl. From the temperature dependence of the rate constants an activation energy of about 60 kJ/mol (at 50 mM NaCl) was estimated, consistent with the hypothesis that a rate limiting step could be the DNA opening frequency. As expected from the stability of a DNA double helix toward large-scale openings, also the activation energy of the strand displacement increases somewhat with increasing salt concentration. There is a nonlinear dependence of the overall rate constant of tripler formation on the PNA concentration with an exponent of 2 or somewhat higher. With PNA in excess, the CD signal shows an overshoot suggesting the presence of a third species. The CD spectrum of this intermediate indicates an ordered structure. The presence of intercalators ethidium bromide and 9-aminoacridine is found to increase the overall PNA binding rate, whereas minor groove binder DAPI and major groove binder methyl green both decrease the rate. The mechanism of strand invasion of duplex DNA by PNA is discussed in the light of these results.

Hoechst 33258 binds with high affinity into the minor groove of AT-rich sequences of double-helical DNA. Despite extensive studies of this and analogous DNA binding molecules, there still remains uncertainty concerning the interactions when multiple ligand molecules are accommodated within close distance. Albeit not of direct concern for most biomedical applications, which are at low drug concentrations, interaction studies for higher drug binding are important as they can give fundamental insight into binding mechanisms and specificity, including drug self-stacking interactions that can provide base-sequence specificity. Using circular dichroism (CD), isothermal titration calorimetry (ITC), and proton nuclear magnetic resonance ((1)H NMR), we examine the binding of Hoechst 33258 to three oligonucleotide duplexes containing AT regions of different lengths: [d(CGCGAATTCGCG)]2 (A2T2), [d(CGCAAATTTGCG)]2 (A3T3), and [d(CGAAAATTTTCG)]2 (A4T4). We find similar binding geometries in the minor groove for all oligonucleotides when the ligand-to-duplex ratio is less than 1:1. At higher ratios, a second ligand can be accommodated in the minor groove of A4T4 but not A2T2 or A3T3. We conclude that the binding of the second Hoechst to A4T4 is not cooperative and that the molecules are sitting with a small separation apart, one after the other, and not in a sandwich structure as previously proposed.

A stretched poly(vinyl alcohol) (PVA) film provides a unique matrix that enables also short DNA oligonucleotide duplex to be oriented and studied by linear dichroism (LD). This matrix further allows controlling DNA secondary structure by proper hydration (A or B form), and such humid films could potentially also mimic the molecular crowding in cellular contexts. However, early attempts to study intercalators and groove binders for probing DNA in PVA failed due to competitive matrix binding. Here we report the successful orientation in PVA of DNA oligonucleotide duplex hairpins with thread-intercalated binuclear complex [μ-(11,11'-bidppz)(phen)4Ru2](4+), and how LD depends on oligonucleotide sequence and metal center chirality. Opposite enantiomers of the ruthenium complex, ΔΔ and ΛΛ, were investigated with respect to enantioselectivity toward GC stretches as long as 22 bp. LD, supported by emission kinetics, reveals that threading intercalation occurs only with ΔΔ whereas ΛΛ remains externally bound, probably in either or both of the grooves of the GC-DNA. Enantioselective binding properties of sterically rigid DNA probes such as the ruthenium complexes could find applications for targeting nucleic acids, e.g., to inhibit transcription in therapeutic context such as treatment of malaria or cancer.

To elucidate the binding mode of recombination protein A (RecA) to double-stranded (ds) DNA, the effects on the RecA-DNA interaction of several mono- and bisintercalators of the acridine, phenanthridine, and cyanine classes have been investigated by linear dichroism spectroscopy. Simple monointercalators lacking side chains efficiently promoted the binding of RecA to dsDNA in the absence of nucleotide cofactor, which is otherwise required. Bisintercalators varied in their ability to induce RecA binding, while monointercalators with aminoalkyl side chains proved inefficient. Modification of DNA structure by the intercalator appears to be necessary for induction of RecA binding, but if the intercalator has a bulky minor-groove-binding side chain, it does not induce RecA binding. In detailed studies with acridines, neither the binding geometry of intercalators nor the structure of DNA was significantly modified upon binding of RecA without cofactor. Judged by circular dichroism, similar RecA conformational changes accompanied bis-9-aminoacridine- and ATP gamma S-induced RecA association with DNA. In the presence of ATP gamma S, the intercalators inhibited the rate of RecA binding to dsDNA and were extruded from DNA upon binding of RecA. This competitive aspect may suggest that intercalation of some amino acid residue(s) plays a role in nucleotide-induced RecA binding. The stoichiometry of the RecA-DNA-intercalator filament was determined; in the fully formed filament the base pair:intercalator ratio is 2, and the base pair:RecA ratio also 2. This contrasts with a base pair:RecA ratio of 3 in the ATP gamma S-induced filament, although in both cases the DNA experiences 50% extension.

The binding of phenanthridine dyes to tripler poly(dT)*poly(dA). poly(dT) and its precursor duplex poly(dA). poly(dT) is characterized using linear dichroism and circular dichroism spectroscopy, and thermal denaturation. The two monomeric dyes ethidium bromide and propidium iodide are shown to behave similarly to each other in intercalating into and stabilizing both the duplex and the tripler structures. However, contrary to expectations, the extra cationic side-chain of propidium iodide provides no significant extra stabilization of tripler compared with ethidium bromide, although propidium does stabilize the duplex more than ethidium. The monomeric dyes appear to have somewhat different binding geometries with the duplex and tripler polymers. The dimeric dye ethidium homodimer is found to bis-intercalate in the tripler as well as the duplex but, in contrast to the monomers, no variation in geometry between duplex and tripler is observed. However, although dimer stabilizes the duplex, it has no effect on the thermal stability of the tripler. This lack of binding preferentiality of the dimer for tripler compared with the monomeric dyes indicates greater constraints on the accommodation of a bis-intercalator in the tripler structure than in the duplex.

Fluorescence and absorption spectroscopy, isothermal titration calorimetry, and viscosity measurements have been used to characterize the interaction of Delta and Lambda [Ru(phen)(2)DPPZ](2+) with calf thymus DNA. The method of continuous variations revealed two distinct binding stoichiometries for both Delta- and Lambda-DPPZ, corresponding to 0.7 and 3 mol of base pair/mol of ligand. Binding isotherms were obtained for the two enantiomers, both of which show strong binding to DNA, with K = 3.2 x 10(6) M(-1) bp and 1.7 x 10(6) M(-1) bp for the Delta and Lambda isomers, respectively, at 25 degrees C in solutions containing 50 mM NaCl. Titration calorimetry gave Delta H values of +0.3 kcal mol(-1) for Delta-DPPZ and +2.9 kcal mol(-1) for Lambda-DPPZ for their interaction with DNA. These small positive enthalpies, which were confirmed using thermal difference spectroscopy, indicated that the binding of these compounds to DNA is entropically driven. An enthalpy of +2.5 kcal mol(-1) was obtained for the binding of the parent compound, tris(phenanthroline)-Ru(II), to DNA. Titration of all three compounds into buffer gave a nonnegligible heat of dilution. The salt dependence of the binding constant was examined for both isomers. The slope SK = (delta logK/delta log[Na+]) was found to be 1.9 and 2.1 for the Delta and Lambda isomers, respectively. By using polyelectrolyte theory to interpret the observed salt dependence of the equilibrium constant, it can be shown that there is a significant nonelectrostatic contribution to the binding constant. Relative viscosity experiments showed that both Delta- and Lambda-DPPZ increase the length of rod-like DNA, in a manner consistent with binding by classical intercalation. Fluorescence energy transfer experiments provided additional evidence for the intercalation of Delta- and Lambda-[Ru(phen)(2)DPPZ](2+) into DNA.

The interactions of RecA with double-stranded DNA and with cofactor adenosine 5'-[3-thio]triphosphate (ATP[S] an analog of ATP) have been characterized by circular dichroism (CD) spectroscopy in a search for conformational changes associated with the formation of helical RecA ATP . DNA . fibers. Upon interaction with the RecA protein the cofactor is found to be structurally perturbed, possibly towards the syn ribose form of ATP[S], while the secondary structure of RecA remains unaffected. By contrast, when the ATP[S] . RecA . DNA complex is formed, a distinct change of the protein CD spectrum near 200 nm is observed as a result of interaction of RecA with DNA. The main change occurs upon the binding of the first DNA molecule [RecA can bind up to three DNA molecules simultaneously; Takahashi, M., Kubista, M. and Norden, B. (1991) Biochimie (Paris) 73, 219-226] and the effect appears to be independent of DNA sequence, suggesting a general change of protein conformation upon DNA binding. The CD of DNA is changed, indicating an alteration of the DNA structure, possibly related to stretching and unwinding. A small, reversible, decrease in the CD amplitude of RecA was observed when raising the temperature from 4 degrees C to 30 degrees C. The CD of RecA increases slightly with pH (up to 7.8) but is constant between pH 6.0 and 6.8. At pH below 6.0 or higher temperature (40 degrees C) slow irreversible denaturation of RecA occurs. The CD signal is effectively independent of salt, even in 2.2 M NaCl or 1 M sodium acetate, which is relevant regarding reported ATPase and coprotease activities promoted by salt. For high concentrations of magnesium (10 mM) at 30 degrees C the CD of RecA changes markedly and the appearance of light scattering indicates aggregation.

Voltage-sensitive dyes are frequently used for probing variations in the electric potential across cell membranes. The dyes respond by changing their spectral properties: measured as shifts of wavelength of absorption or emission maxima or as changes of absorption or fluorescence intensity. Although such probes have been studied and used for decades, the mechanism behind their voltage sensitivity is still obscure. We ask whether the voltage response is due to electrochromism as a result of direct field interaction on the chromophore or to solvatochromism, which is the focus of this study, as result of changed environment or molecular alignment in the membrane. The spectral properties of three styryl dyes, di-4-ANEPPS, di-8-ANEPPS, and RH421, were investigated in solvents of varying polarity and in model membranes using spectroscopy. Using quantum mechanical calculations, the spectral dependence of monomer and dimer ANEPPS on solvent properties was modeled. Also, the kinetics of binding to lipid membranes and the binding geometry of the probe molecules were found relevant to address. The spectral properties of all three probes were found to be highly sensitive to the local environment, and the probes are oriented nearly parallel with the membrane normal. Slow binding kinetics and scattering in absorption spectra indicate, especially for di-8-ANEPPS, involvement of aggregation. On the basis of the experimental spectra and time-dependent density functional theory calculations, we find that aggregate formation may contribute to the blue-shifts seen for the dyes in decanol and when bound to membrane models. In conclusion, solvatochromic and other intermolecular interactions effects also need to be included when considering electrochromic response voltage-sensitive dyes.

The possibility that the stacked DNA bases can mediate vectorial electron transfer has been examined using two different approaches. Experiments on photoinduced electron transfer with intercalated donors and acceptors (either randomly bound or linked dyads of ruthenium complex and viologen) indicate that while DNA may be a better medium than acetonitrile for electron transfer over short distances (2-3-base pair, equivalent to 10-14Å centre-to-centre separation), it is a poor medium for transport over larger separations. Attempts to measure conductivity of individual DNA molecules using scanning tunneling microscopy to image mixed monolayers of mercaptohexanol (MCH) and 30-mer or 10-mer DNAs with alkanethiol linkers also indicate that DNA in its native state is a poor conductor. AFM images of the DNA/MCH mixed monolayers show that the DNA molecules extend vertically upward from the surface in such surface architectures.

Opposite enantiomers of [Ru(phenanthroline)(3)](2+) affect the persistence length of DNA differently, a long speculated effect of helix kinking. Our molecular dynamics simulations confirm a substantial change of duplex secondary structure produced by wedge-intercalation of one but not the other enantiomer. This effect is exploited by several classes of DNA operative proteins.

In various chemical systems enthalpy-entropy compensation (EEC) is a well-known rule of behavior, although the physical roots of it are still not completely understood. It has been frequently questioned whether EEC is a truly physical phenomenon or a coincidence due to trivial mathematical connections between statistical-mechanical parameters - or even simpler: A phantom effect resulting from the misinterpretation of experimental data. Here, we review EEC from a new standpoint using the notion of correlation which is essential for the method of factor analysis, but is not conventional in physics and chemistry. We conclude that the EEC may be rationalized in terms of hidden (not directly measurable with the help of the current experimental set-up) but physically real factors, implying a Carnot-cycle model in which a micro-phase transition (MPT) plays a crucial role. Examples of such MPTs underlying physically valid EEC should be typically cooperative processes in supramolecular aggregates, like changes of structured water at hydrophobic surfaces, conformational transitions upon ligand-biopolymer binding, and so on, so forth. The MPT notion could help rationalize the occurrence of EEC in connection with hydration and folding of proteins,enzymatic reactions, functioning of molecular motors, DNA de- and rehybridization, as well as similar phenomena., 8 pages, 2 Figures, Submitted for publication

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

Desorption electrospray ionization (DESI) coupled to high-resolution Orbitrap mass spectrometry (MS) was used to study the reactivity of a (β-amino alcohol)(arene)RuCl transfer hydrogenation catalytic precursor in methanol (CH3OH). By placing [(p-cymene)RuCl2]2 on a surface and spraying a solution of β-amino alcohol in methanol, two unique transient intermediates having lifetimes in the submillisecond to millisecond range were detected. These intermediates were identified as Ru (II) and Ru (IV) complexes incorporating methyl formate (HCOOCH3). The Ru (IV) intermediate is not observed when the DESI spray solution is sparged with Ar gas, indicating that O2 dissolved in the solvent is necessary for oxidizing Ru (II) to Ru (IV). These proposed intermediates are supported by high-resolution and high mass accuracy measurements and by comparing experimental to calculated isotope profiles. Additionally, analyzing the bulk reaction mixture using gas chromatography-MS and nuclear magnetic resonance spectroscopy confirms the formation of HCOOCH3. These results represent an example that species generated from the (β-amino alcohol)(arene)RuCl (II) catalytic precursor can selectively oxidize CH3OH to HCOOCH3. This observation leads us to propose a pathway that can compete with the hydrogen transfer catalytic cycle. Although bifunctional hydrogen transfer with Ru catalysts has been well-studied, the ability of DESI to intercept intermediates formed in the first few milliseconds of a chemical reaction allowed identification of previously unrecognized intermediates and reaction pathways in this catalytic system.

We report remarkable multiphoton absorption properties of DNA intercalating ruthenium complexes: (1) [Ru(phen)(2)dppz](2+); (2) [(11,11'-bidppz)(phen)(4)Ru(2)](4+); (3) [11,11'-bipb(phen)(4)Ru(2)](4+). Two-photon spectra in the range from 460 to 1100 nm were measured using the Z-scan technique. In particular, complex 2 was found to exhibit very strong two- and three-photon absorption properties, which could be an effect of symmetric charge transfer from the ends towards the middle of the conjugated dimeric orbital system. We propose that these molecules could provide a new generation of DNA binding nonlinear chromophores for wide applications in biology and material science. The combination of a large two-photon cross section and strong luminescence quantum yields for the molecules when intercalated makes the compounds uniquely bright and photo-stable probes for two-photon luminescence imaging and also promising as enhanced photosensitizers in two-photon sensitizing applications.

Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved internalization, we used flow cytometry to examine uptake in relation to cell surface binding for penetratin and two arginine/lysine substituted variants (PenArg and PenLys) in wildtype CHO-K1 and PG-deficient A745 cells. All peptides were more efficiently internalized into CHO-K1 than into A745, but their cell surface binding was independent of cell type. Thus, PGs promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Uptake of each peptide was linearly dependent on its cell surface binding, and affinity is thus important for efficiency. However, the gradients of these linear dependencies varied significantly. Thus each peptide's ability to stimulate uptake once bound to the cell surface is reliant on formation of specific uptake-promoting interactions. Heparin affinity chromatography and clustering experiments showed that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts through electrostatic attraction. This may have implications for the molecular mechanisms behind arginine-specific uptake stimulation as penetratin and PenArg are more efficiently internalized than PenLys upon interaction with PGs. However, PenArg is also least affected by removal of PGs. This indicates that an increased arginine content not only improve PG-dependent uptake but also that PenArg is more adaptable as it can use several portals of entry into the cell.

The Z-scan technique was used to determine the spectral dependence of the nonlinear absorption in well-known DNA intercalators: ethidium bromide and its homodimer. It is found that the compounds show essentially the same features of their nonlinear absorption spectra with the magnitudes of the relevant cross sections scaling with molecular weight of chromophore compound.

Catalysts are agents that by binding reactant molecules lower the energy barriers to chemical reaction. After reaction the catalyst is regenerated, its unbinding energy recruited from the environment, which is associated with an inevitable loss of energy. We show that combining several catalytic sites to become energetically and temporally phase-shifted relative to each other provides a possibility to sustain the overall reaction by internal 'energy recycling', bypassing the need for thermal activation, and in principle allowing the system to work adiabatically. Using an analytical model for superimposed, phase-shifted potentials of F-1-ATP synthase provides a description integrating main characteristics of this rotary enzyme complex.

Cell-penetrating peptides and antimicrobial peptides are two classes of positively charged membrane active peptides with several properties in common. The challenge is to combine knowledge about the membrane interaction mechanisms and structural properties of the two classes to design peptides with membrane-specific actions, useful either as transporters of cargo or as antibacterial substances. Membrane active peptides are commonly rich in arginine and tryptophan. We have previously designed a series of arg/trp peptides and investigated how the position and number of tryptophans affect cellular uptake. Here we explore the antimicrobial properties and the interaction with lipid model membranes of these peptides, using minimal inhibitory concentrations assay (MIC), circular dichroism (CD) and linear dichroism (LD). The results show that the arg/trp peptides inhibit the growth of the two gram positive strains Staphylococcus aureus and Staphylococcus pyogenes, with some individual variations depending on the position of the tryptophans. No inhibition of the gram negative strains Proteus mirabilis or Pseudomonas aeruginosa was noticed. CD indicated that when bound to lipid vesicles one of the peptides forms an α-helical like structure, whereas the other five exhibited rather random coiled structures. LD indicated that all six peptides were somehow aligned parallel with the membrane surface. Our results do not reveal any obvious connection between membrane interaction and antimicrobial effect for the studied peptides. By contrast cell-penetrating properties can be coupled to both the secondary structure and the degree of order of the peptides.