CO2 can be enzymatically reduced to methanol in a cascade reaction involving three enzymes: formate-, formaldehyde- and alcohol dehydrogenase (FateDH, FaldDH, ADH).

The 2,6,10-tris(dialkylamino)trioxatriangulenium dyes (ATOTA+) are highly stabilised cationic chromophores with D3h symmetry. The symmetry gives rise to a degeneracy of the main electronic transition. In low polarity solvents significant splitting of this degenerate transition is observed and assigned to ion pair formation. Ion pairing of the 2,6,10-tris(dioctylamino)trioxatriangulenium ion with Cl−, BF4−, PF6− and TRISPHAT anions was studied using absorption spectroscopy. A clear correlation is found between the size of the anion and the splitting of the ATOTA+ transitions. In benzene the Cl− salt displays a splitting of 1955 cm−1, while the salt of the much larger TRISPHAT ion has a splitting of 1543 cm−1. TD-DFT calculations confirm the splitting of the states and provide a detailed insight into the electronic structure of the ion pairs. The different degree of splitting in different ion pairs is found to correlate with the magnitude of the electric field generated in each ion pair, thus leading to the conclusion that the effect seen is an internal Stark effect. By insertion of an amphiphilic derivative of the ATOTA+ chromophore in an oriented lamellar liquid crystal, it was possible to resolve the two bands of the double peak spectrum and show their perpendicular orientation in the molecular framework, as predicted by the calculations.

Mesoporous silica (MPS) particles are promising materials for the immobilization of a variety of enzymes due to the possibility of tailoring their morphology and surface properties. A better understanding of the spatial distribution of enzymes immobilized in these materials is important for improved biocatalytic applications. In this work three types of MPS materials are compared for the immobilization of alcohol dehydrogenase (ADH): two ordered mesoporous silica type SBA-15 particles with 6.8 and 10 nm pore diameters and one mesostructured cellular foam (MCF) with a pore diameter of 26.8 nm and window diameter of 10.7 nm. Nitrogen sorption analysis and transmission electron microscopy (TEM) using immunogold staining (IGS) are used to study the immobilized amount and spatial . distribution of ADH in the MPS. In the MCF the mesopore volume occupied by the enzymes measured by nitrogen sorption analysis agrees well with the calculated pore filling, which indicates that the amount of enzyme bound to the external surface is small as supported by TEM imaging showing an essentially even distribution in the MCF particles at least up to 2000 nm from the particle surface. In the case of SBA-15 with 6.8 nm pore size the ADH can enter the pores even though the pore size distribution is smaller than the hydrodynamic size of the protein, but the enzymes only penetrate to about 500 nm from the particle surface. With 10 nm pores the ADH penetrates further into SBA-15 but is still not evenly distributed. MCF can accommodate the largest amount of enzyme per gram of particles and still has a substantial fraction of its mesopore volume available, showing that MCF is a good host for the immobilization of large enzymes such as ADH.

Fluorescence spectroscopy of protein-bound molecular rotors Cy3 and Cy5 is used to monitor the effective viscosity inside the pores of two types of mesoporous silica (SBA-15 and MCF) with pore diameters between 8.9 and 33 nm. The ratio of the peak intensities is used to measure viscosity independently of solvent polarity, and the response of the lipase-bound dyes is calibrated using glycerol/water mixtures (no particles). The two dyes are either attached to the same protein or separate proteins in order to investigate potential effects of energy transfer (FRET) on the fluorescence properties, when using them as reporter dyes. The effective viscosity inside the pores at infinite protein dilution is one order of magnitude higher than in bulk water, and the effect of protein concentration on the measured viscosity indicates a stronger effect of protein-protein interactions in the pores than in similarly concentrated protein solutions without particles. In MCF-particles with octyl-groups covalently attached to the pore walls, a more efficient uptake of the lipase resulted in FRET between the protein-bound dyes even if the two dyes were attached to different proteins. In contrast to the unmodified particles the intensity-ratio method could therefore not be used to measure the viscosity, but the presence of FRET in itself indicates that octyl-protein interactions lead to a non-homogenous protein distribution in the pores. The dye labels also report a less polar pore environment as sensed by the proteins through a redshift in the dye emission. Both observations may help in understanding the higher efficiency of lipase immobilization in octyl-modified particles.

By combining two enzymes, formate dehydrogenase (FateDH) and formaldehyde dehydrogenase (FaldDH), it is possible to drive the thermodynamically unfavorable conversion of CO2 to formaldehyde. For this purpose, the enzymes were coimmobilized in siliceous mesostructured cellular foams (MCFs). A high degree of adsorption of both enzymes was achieved by coimmobilizing the enzymes sequentially, i.e., first FateDH and then FaldDH. The highest conversion rate was obtained with an enzyme mass ratio of 1:15 (FateDH/FaldDH). Using MCF functionalized with mercaptopropyl groups (MCF-MP), the activity increased similar to 4 times compared to the enzymes free in solution. To probe the distance between the two enzymes, they were separately labeled with either Cy3 or Cy5 dyes and studied with Forster resonance energy transfer (FRET). An increased energy transfer was observed when the enzymes were coimmobilized in MCF-MP, suggesting that the two enzymes are in close proximity, resulting in higher conversion of CO2 to formaldehyde.

Interaction of double stranded DNA with bulky and hydrophobic Salen type Schiff base complex: [N, N' Bis [3- tert-butyl-5-[triphenyl-phosphonium - methyl] - salicylidene] 1,2 ethylene-diamine nickel(III) acetate (refer to Ni Salen complex) was extensively investigated using the spectroscopic techniques and gel electrophoresis. Absorption titration experiment showed the hypochromic effect and the significant red shift of the complex absorption. In competition experiments with ethidium bromide (EB), Ni Salen complex exhibited non-competitive binding at high concentrations. UV-vis absorption and fluorescence emission data agreed on a binding constant of (1.64 ± 0.01) μM-1, thereby showing the strong interaction of the complex with DNA; also, a binding site size of 2.33 ± 0.01 base pairs per complex was achieved. Thermal denaturation experiment showed that Tm of calf thymus-DNA was increased by approximately 10 °C at a molar ratio of the dye/base of 0.2. The CD spectra of DNA exhibited an increase in both positive and negative peaks without any shift in the position of bands upon addition of the complex. The amplitude of the LD spectra of DNA was decreased in the presence of the complex. Reduced linear dichroism (LDred) revealed that the transition moment of complex was parallel to the DNA helix axis. Gel electrophoresis experiments confirmed that Ni Salen complex had no nuclease/DNA cleaving activity; also, DNA-DNA cross links were formed at high concentrations of complex, leading to the aggregation of DNA.

Enzyme immobilization in porous silica particles is used to improve enzyme function in biocatalytic applications. Here, we study the effective protein concentration and rotational mobility of lipase and bovine serum albumin in the pores, when confined in five types of mesoporous silica particles with different pore and particle sizes, exploiting the intrinsic UV-vis absorption and fluorescence anisotropy of the tryptophan residues. For all investigated combinations of proteins and particles, the steady-state anisotropy is higher than for the same protein in free solution, indicating a slower protein rotation inside the pores. The retardation is stronger in more narrow pores, but the proteins can still move, and there is no dependence on the particle size. The average number of proteins per particle, Nprot, varies with the particle diameter, D, as Nprot ∼ D2.95±0.02 for both proteins, which is close to the scaling D3.0±0.1 for the available pore volume. This observation indicates that both proteins are distributed evenly throughout the particles and rules out that the proteins are only externally bound to the particle surface. Secondly, the concentration of the protein in the pores depends on the pore and protein size but not on the particle size and corresponds to volume fractions in the range of 20-60%.

Mesoporous silica particles are used for immobilization of enzymes in order to increase enzyme stability, facilitate product purification and reuse of enzyme. Many efforts have been done to investigate how the environment inside the pores changes after enzyme immobilization and how it can affect activity and stability of immobilized enzymes. One of these environmental changes can be viscosity inside the pores and comparing the difference with the bulk solution. Here, we investigate viscosity inside mesoporous silica particles, SBA-15, 1µm diameter with 9nm pore diameter, based on two different cyanine fluorophores which are sensitive to viscosity, Cy3 and Cy5 using a ratiometric method. The probes can bond covalently to lipase as enzyme used in two different conditions: a. both probes seated on one enzyme, b. they can be attached to the enzyme separately. We work with a ratiometric method to measure only viscosity and no other solvent effects. In this study, Cy5 can be used to normalize for polarity other than viscosity. in addition, we take advantage of the energy transfer (FRET) between probes when they are seated on one enzyme and measure the distance between them.

Mesoporous silica particles are used for immobilization of enzymes in order to increase enzyme stability, facilitate product purification and reuse of enzyme. An important question is how the immobilized enzymes distribute on the surface or inside the pores of silica particles, the difference between porous particles and non-porous particles can be helpful to investigate the effect of pores on the capacity of loading for the particles. Here, we examine the immobilization of lipase in three different types of mesoporous silica particles with different pore size but the same diameter, 5 µm. The hydrodynamic radius of lipase id 2.25 nm; while the radius of the silica particle pores is 12nm, 6nm and 2nm. In this research secondary ion mass spectroscopy (SIMS) is used as a novel method to monitor the distribution of immobilized enzymes. Also high resolution fluorescence microscopy is used to compare with SIMS results. The immobilization is followed by recording the fluorescence from epicocconone, a dye binding to the enzyme.

Mesoporous silica particles are used as support material for immobilization of enzymes. Here we investigated a fluorescence-based assay for real-time monitoring of the immobilization of lipase, bovine serum albumin, and glucose oxidase into micrometer-sized mesoporous silica particles. The proteins are labeled with the dye epicocconone, and the interaction with the particles is observed as an increase in emission intensity of the protein–dye conjugates that can be quantified if correcting for a comparatively slow photobleaching. The immobilization occurs in tens of minutes to hours depending on particle concentration and type of protein. In the limit of excess particles over proteins, the formation of the particle–protein complexes can be described by a single exponential growth for all three investigated proteins, and the fitted pseudo-first-order rate constant increases linearly with particle concentration for each protein type. The derived second-order rate constant k varies with the protein hydrodynamic radius according to k∼RH−4.70±0.01, indicating that the rate-limiting step at high particle concentrations is not the diffusional encounter between proteins and particles but rather the entry into the pores, consistent with the hydrodynamic radii of the three proteins being smaller but comparable to the pore radius of the particles.

Biosensors, in which binding of ligands is detected through changes in the optical or electrochemical properties of a DNA layer confined to the sensor surface, are important tools for investigating DNA interactions. Here, we investigate if conformational changes induced in surface-attached DNA molecules upon ligand binding can be monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. DNA duplexes containing 59-184 base pairs were formed on QCM-D crystals by stepwise assembly of synthetic oligonucleotides of designed base sequences. The DNA films were exposed to the cationic polyamines spermidine and spermine, known to condense DNA molecules in bulk experiments, or to the recombination protein Rad51, known to extend the DNA helix. The binding and dissociation of the ligands to the DNA films were monitored in real time by measurements of the shifts in resonance frequency (Δf) and in dissipation (ΔD). The QCM-D data were analyzed using a Voigt-based model for the viscoelastic properties of polymer films in order to evaluate how the ligands affect thickness and shear viscosity of the DNA layer. Binding of spermine shrinks all DNA layers and increases their viscosity in a reversible fashion, and so does spermidine, but to a smaller extent, in agreement with its lower positive charge. SPR was used to measure the amount of bound polyamines, and when combined with QCM-D, the data indicate that the layer condensation leads to a small release of water from the highly hydrated DNA films. The binding of Rad51 increases the effective layer thickness of a 59 bp film, more than expected from the know 50% DNA helix extension. The combined results provide guidelines for a QCM-D biosensor based on ligand-induced structural changes in DNA films. The QCM-D approach provides high discrimination between ligands affecting the thickness and the structural properties of the DNA layer differently. The reversibility of the film deformation allows comparative studies of two or more analytes using the same DNA layer as demonstrated here by spermine and spermidine.

Mesoporous silica particles are used for immobilization of enzymes in order to increase enzyme stability, facilitate product purification and reuse of enzyme. An important question is how the size of the enzyme affects the rate of immobilization and also whether the immobilization rate depends on silica particle concentration. Here, we examine the immobilization of three enzymes with different size. The enzymes used are Lipase, Bovine serum albumin (BSA), Glucose oxidase (Gox) and the diameter of them, respectively, are 4nm, 7nm, and 9nm; while the diameter of the silica particle pores is 9nm. In this research fluorescence spectroscopy is used as a direct monitoring technique. The immobilization is followed by recording the fluorescence from epicocconone, a dye binding to the enzyme. The results show that there is a relation between the size of enzyme and the immobilization rate, the larger the enzyme the slower the rate of immobilization. Also the investigation shows that by increasing mesoporous silica concentration the immobilization rate increases. Further, real-time data on immobilization rate and the enzyme and particle concentration dependence can be used to test models for the immobilization process.

Studies of DNA–ligand interaction on a single molecule level provide opportunities to understand individual behavior of molecules. Construction of DNA molecules with repetitive copies of the same segments of sequences linked in series could be helpful for enhancing the interaction possibility for sequence-specific binding ligand to DNA. Here we report on the use of synthetic oligonucleotides to self-assembly into duplex DNA concatemeric molecules. Two strands of synthetic oligonucleotides used here were designed with 50-mer in length and the sequences are semi-complimentary so to hybridize spontaneously into concatemers of double stranded DNA. In order to optimize the length of the concatemers the oligonucleotides were incubated at different oligomer concentrations, ionic strengths and temperatures for different durations. Increasing the salt concentration to 200mM NaCl was found to be the major optimizing factor because at this enhanced ionic strength the concatemers formed most quickly and the other parameters had no detectable effect. The size and shape of formed DNA concatemers were studied by gel electrophoresis in agarose, polyacrylamide gels and by AFM. Our results show that linear DNA constructs up to several hundred base pairs were formed and could be separated from a substantial fraction of non-linear constructs.

The sequential hybridization of a 534 base pair DNA concatemer layer was monitored by QCM-D and SPR, and the QCM-D data were analyzed by Voigt viscoelastic models. The results show that Voigt-based modeling gives a good description of the experimental data but only if shear viscosity and elasticity are allowed to depend on the shear frequency. The derived layer thickness, shear viscosity and elasticity of the growing film give a representation of the DNA film in agreement with known bulk properties of DNA, and reveal a maximum in film viscosity when the molecules in the layer contain 75 base pairs. The experimental data during construction of a 3084 bp DNA concatemer layer were compared to predictions of the QCM-D response of a 1 μm thick film of rod-like polymers. A predicted nonmonotonous variation of dissipation with frequency (added mass) is in qualitative agreement with the experiments, but with a quantitative disagreement which likely reflects that the flexibility of such long DNA molecules is not included in the model.

Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at the molecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials.

Overstretching of DNA occurs at about 60-70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.

842-Pos Board B611 Single-Molecule Assessment of DNA Supercoiling and Relaxation by S. Typhimurium and E. Coli DNA Gyrases Mónica Fernández-Sierra, Chandler H. Fountain, Laura Finzi, David D. Dunlap. Emory University, Atlanta, GA, USA. Type II topoisomerases maintain DNA topology by regulating the level of supercoiling of chromosomes. DNA gyrase is a unique and highly conserved bacterial Type II topoisomerase which is able to introduce negative supercoils into the genome. Using magnetic-tweezers, we assessed the supercoil generation and relaxation activities of Escherichia coli and Salmonella typhimurium DNA gyrases. Our results indicate that under single-enzyme conditions and 0.6-pN tension, both enzymes relax DNA at similar rates, but Salmonella gyrase pauses more often. At high enzyme concentration and lower tensions, Salmonella gyrase introduced negative supercoils faster and to a larger extent than E. coli gyrase. Sequence and structure analyses show that most of the differences between the two enzymes are in the C-terminal domain, involved in DNA wrapping. Our ongoing single-molecule experiments to assess the DNA wrapping activities of both enzymes may reveal differences in their mechanistic properties.

The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.

We report that DNA molecules can be intercalated and macroscopically oriented in the aqueous interstitia of a lyotropic lamellar liquid crystal. Using UV-vis linear dichroism and fluorescence spectroscopy we show that double-stranded oligonucleotides (25 base pairs) in the water-octanoate-decanol system remain base-paired in the B conformation and are confined in two dimensions, with the helix axis preferentially parallel to the lipid bilayer surfaces but free to rotate within this plane. The degree of helix confinement and the corresponding 2-D orientation can be improved by decreasing the thickness of the water interstitia via the fraction of water in the ternary mixture. Not surprisingly, the corresponding single-stranded oligonucleotides are not aligned, with their persistence length being short in comparison to the lamellar interstitium thickness. We propose this as a model system for studying interactions of DNA-ligand complexes near a lipid bilayer membrane which we demonstrate by using dye probes that are either covalently attached to one end of the oligonucleotide or reversibly bound by intercalation between the base pairs. Three cationic dyes, all strongly bound by intercalation to DNA when free in solution, are found to not bind to DNA but to prefer the membrane surface. The covalently attached Cy5 also binds to the bilayer while Cy3 tends to end-stack to the oligonucleotide duplex. The orientation of Cy5 parallel to the membrane indicates that electrostatic surface binding predominates over insertion into the hydrophobic interior of the membrane. Anionic and zwitterionic dyes (FAM and ROX) are found to remain randomly oriented in the water between the lipid bilayer surfaces.

Mixed-sequence DNA molecules undergo mechanical overstretching by approximately 70% at 60-70 pN. Since its initial discovery 15 y ago, a debate has arisen as to whether the molecule adopts a new form [Cluzel P, et al. (1996) Science 271: 792-794; Smith SB, Cui Y, Bustamante C (1996) Science 271: 795-799], or simply denatures under tension [van Mameren J, et al. (2009) Proc Natl Acad Sci USA 106: 18231-18236]. Here, we resolve this controversy by using optical tweezers to extend small 60-64 bp single DNA duplex molecules whose base content can be designed at will. We show that when AT content is high (70%), a force-induced denaturation of the DNA helix ensues at 62 pN that is accompanied by an extension of the molecule of approximately 70%. By contrast, GC-rich sequences (60% GC) are found to undergo a reversible overstretching transition into a distinct form that is characterized by a 51% extension and that remains base-paired. For the first time, results proving the existence of a stretched basepaired form of DNA can be presented. The extension observed in the reversible transition coincides with that produced on DNA by binding of bacterial RecA and human Rad51, pointing to its possible relevance in homologous recombination.