Active uptake of drugs into photosensitive liposomes and rapid release on UV photolysis

Liposomes containing high concentrations of the anticancer drug doxorubicin, prepared by active-loading techniques, have been intensively investigated as potential agents for chemotherapy. The present study investigates the possibility of active uptake and photoinduced release of such solutes from liposomes incorporating a photoisomerizable lipid. The active loading of acridine orange and doxorubicin was investigated using liposomes containing entrapped ammonium sulfate. The liposomes were prepared with dipalmitoyl-L-alpha-phosphatidyl choline (DPPC) and a photochromic lipid, (1,2-(4'-n-butylphenyl)azo-4'-(gamma-phenylbutyroyl))-glycero-3- phosphocholine (Bis-Azo PC), which isomerizes on exposure to near-UV light with resulting changes in membrane permeability to solutes. The rate of loading of the vesicles below the phase transition temperature of DPPC was investigated as a function of Bis-Azo PC and cholesterol concentrations in the liposome. The rate of doxorubicin uptake was found to be greatly decreased in the presence of cholesterol, while below 30 degrees C the rate of acridine orange uptake was increased in the presence of cholesterol. On exposure to a single UV laser pulse, actively loaded acridine orange was rapidly released from liposomes containing Bis-Azo PC at a rate similar to that found for the indicator dye calcein. However while cholesterol had previously been shown to greatly enhance the rate of photo-induced calcein leakage, it had no significant effect on the rate of acridine orange release. After active loading into DPPC vesicles containing Bis-Azo PC, doxorubicin was also released after exposure to a single laser pulse, but at a rate slower than for acridine orange and calcein. The difference in behavior between these systems is ascribed to the interactions of acridine orange and doxorubicin with the liposome bilayer. Photoinduced release of pharmacologically active materials from sensitized liposomes might provide a useful adjunct or alternative to conventional photodynamic therapy.     

Self‐Assembled Multilayers of Polyethylenimine and DNA: Spectrophotometric and Electrochemical Characterization and Application for the Determination of Acridine Orange Interaction

This work deals with the study of the interaction between acridine orange (AO) and calf‐thymus double stranded DNA (dsDNA) present in supramolecular architectures built on gold electrodes modified with mercapto‐1‐propanesulfonic acid (MPS) by self‐assembling of polyethylenimine and dsDNA. The optimal conditions for building the supramolecular architecture were obtained from UV‐vis spectrophotometric experiments. The electrochemical studies were performed by adsorptive transfer square wave voltammetry from the evaluation of the oxidation signal of AO accumulated within the multistructure. The effect of the number of PEI‐dsDNA bilayers (Au/MPS/(PEI‐dsDNA)_n) on the accumulation and electrooxidation of AO is also discussed.     

Electrochemical DNA Biosensors Applicable to the Study of Interactions Between DNA and DNA Intercalators

Electrochemical DNA biosensors, based either on carbon paste electrode (CPE) or hanging mercury drop electrode (HMDE) were prepared. These biosensors were used in the study of interaction between double stranded DNA (dsDNA) and single stranded DNA (ssDNA) and acridine orange, a well known DNA intercalator. The different electrochemical behaviors were compared in the article.     

Probing the mechanical stability of DNA in the presence of monovalent cations

We examine the interaction between monovalent cations and DNA using several different assays that measure the stability of double-stranded DNA (dsDNA). The thermal melting of dsDNA and the mechanical separation of dsDNA into two single strands both depend on the stability of dsDNA with respect to ssDNA and are sensitive to the interstrand phosphate repulsion. We find that the experimentally measured melting temperatures and unzipping forces are approximately the same for all of the ions considered in this study. Likewise, the force required to transform B-DNA into the overstretched form is also similar for all of the ions. In contrast, for a given ion concentration, the force at which the overstretched state fully relaxes back to the canonical B-DNA form depends on the cation; however, for all cations, the overstretching force decreases with decreasing ion concentration, suggesting that this force is sensitive to screening. We observe a general trend for smaller ions to produce more efficient relaxation. Finally, for a given cation, the relaxation can also depend on the anion.     

Synthetic dsDNA‐Binding Peptides Using Natural Compounds as Model

We have developed a series of short DNA‐binding peptides containing newly synthesized, unnatural as well as natural amino acid building blocks. By a combinatorial‐library approach, oligopeptides were developed with moderate dsDNA‐binding affinities. Two strategies were used to further enhance the binding affinity of the lead peptides: Ac‐Arg‐Ual‐Sar‐Chi‐Chi‐Chi‐Arg‐NH₂ and Ac‐Arg‐Cbg‐Cha‐Chi‐Chi‐Tal‐Arg‐NH₂. Site‐selective amino acid substitutions increased the binding affinities up to 2 × 10^?5 M . Further enhancement of the binding affinities could be achieved by coupling of an acridine intercalating unit, using linker arms of different length and flexibility. With the introduction of a new lysine‐based acridine unit, different types of oligopeptide–acridine conjugates were designed using known dsDNA‐binding ligands as model compounds. The binding capacities of these new oligopeptide–acridine conjugates have been investigated by a fluorescent intercalator (ethidium bromide) displacement (FID) assay. With the synthesis of the dipeptide–acridine conjugates, binding affinities in the low micromolar range were obtained (6.4 × 10^?6 M ), which is similar to the binding strength of the well‐known DNA binder Hoechst 33258.     

Cyclic voltammetric detection of chemical DNA damage induced by styrene oxide in natural dsDNA layer-by-layer films using methylene blue as electroactive probe

In this work, an electrochemical sensor for detecting damage of natural double-stranded DNA (dsDNA) was constructed based on PSS/{PDDA/dsDNA}₃ layer-by-layer films, where PSS was poly(styrene sulfonate) and PDDA was poly(diallyldimethyl ammonium). When the PSS/{PDDA/dsDNA}₃ films assembled on pyrolytic graphite (PG) electrodes were immersed into methylene blue (MB) solution and loaded MB into the films, the formed PSS/{PDDA/dsDNA}₃-MB films in blank buffers at pH 7.0 showed a reversible cyclic voltammetric (CV) peak pair at −0.23 V vs. SCE for MB redox couple. In blank solutions, the MB loaded into the films was released gradually from the films, but the complete reloading of MB into the films could be realized by immersing the films into MB solution again, indicating the good reversibility of MB incorporation. However, after incubation in the solution of known genotoxic agent styrene oxide (SO), the damaged PSS/{PDDA/dsDNA}₃-MB films could not return to their original and fully-loaded state with reloading of MB, and showed smaller CV peak currents than those of intact PSS/{PDDA/dsDNA}₃-MB films. The relative peak current ratio, I_p,I/I_p,III, where I_p,I was the anodic peak current of intact DNA films in blank buffers after fully loading with MB and I_p,III was that of SO-damaged DNA films after reloading with MB, increased linearly with the incubation time with SO in the range of 5–40 min with a damage rate of 0.0099 min⁻¹. While the formation of SO adducts with dsDNA had no substantial effect on the dsDNA conformation, the steric hindrance of SO adducts with guanine or adenine blocked the intercalation of MB into the base pairs of dsDNA, resulting in the decrease of CV peak currents of loaded MB. The specific intercalation of MB into dsDNA base pairs and the sensitive electrochemical response of MB, combined with the unique feature of loading reversibility of MB in the DNA layer-by-layer films, make the difference in CV response between the intact and damaged dsDNA films become pronounced in the “loading/release/reloading” procedure. This may provide a new and general approach for constructing a biosensor for screening genetoxic chemicals in vitro.     

In situ evaluation of anticancer drug methotrexate-DNA interaction using a DNA-electrochemical biosensor and AFM characterization

An in situ evaluation of the dsDNA-methotrexate (MTX) interaction was performed by voltammetry using a DNA-electrochemical biosensor and characterized by atomic force microscopy (AFM) at a highly oriented pyrolytic graphite (HOPG) surface. Electrochemical experiments in incubated solutions showed that the interaction of MTX with dsDNA leads to modifications to the dsDNA structure in a time-dependent manner. The AFM images show reorganization of the DNA self-assembled network on the surface of the HOPG electrode upon binding methotrexate and the formation of a more densely packed and slightly thicker MTX-dsDNA lattice with a large number of aggregates embedded into the network film. The intercalation of MTX between complementary base pairs of dsDNA lead to the increase of purine oxidation peaks due to the unwinding of the dsDNA. The dsDNA-electrochemical biosensor and the purinic homo-polynucleotide single stranded sequences of guanosine and adenosine, poly[G] and poly[A]-electrochemical biosensors, were used to investigate and understand the interaction between MTX and dsDNA.     

Cytosine methylation regulates DNA bendability depending on the curvature

Cytosine methylation plays an essential role in many biological processes, such as nucleosome inactivation and regulation of gene expression. The modulation of DNA mechanics may be one of the regulatory mechanisms influenced by cytosine methylation. However, it remains unclear how methylation influences DNA mechanics. Here, we show that methylation has contrasting effects on the bending property of dsDNA depending on DNA curvature. We directly applied bending force on 30 base pairs of dsDNA using a D-shaped DNA nanostructure and measured the degree of bending using single-molecule fluorescence resonance energy transfer without surface immobilization. When dsDNA is weakly bent, methylation increases the stiffness of dsDNA. The stiffness of dsDNA increased by approximately 8% with a single methylation site for 30 bp dsDNA. When dsDNA is highly bent by a strong force, it forms a kink, i.e., a sharp bending of dsDNA. Under strong bending, methylation destabilizes the non-kink form compared with the kink form, which makes dsDNA near the kink region apparently more bendable. However, if the kink region is methylated, the kink form is destabilized, and dsDNA becomes stiffer. As a result, methylation increases the stiffness of weakly bent dsDNA and concurrently can promote kink formation, which may stabilize the nucleosome structure. Our results provide new insight into the effect of methylation, showing that cytosine methylation has opposite effects on DNA mechanics depending on its curvature and methylation location.

D-shaped DNA is used to observe dsDNA bending mechanics. Cytosine methylation increases the intrinsic stiffness of dsDNA. Under strong bending, methylation stabilizes or destabilizes a kink form depending on methylation sites.     

Interaction between dendrons directly studied by single-molecule force spectroscopy

In this article, we have investigated the interaction between two poly(benzyl ether) dendrons directly by single-molecule force spectroscopy. For this purpose, one dendron was immobilized on an AFM tip through a poly(ethylene glycol) (PEG) spacer, and the other dendron was anchored on a gold substrate as a self-assembled monolayer. Two dendrons approached and then interacted with each other when the AFM tip and the substrate moved close together. The rupture force between dendrons was measured while the AFM tip and the substrate separated. PEG as a flexible spacer can function as a length window for recognizing the force signals and avoiding the disturbance of the interaction between the AFM tip and the substrate. The interaction between two first-generation dendrons is measured to be about 224 pN at a force loading rate of 40 nN/s. The interaction between second- and first-generation dendrons rises to 315 pN at the same loading rate. Such interactions depend on the force loading rate in the range of several to hundreds of nanonewtons per second, indicating that the rupture between dendrons is a dynamic process. The study of the interaction between surface-bound dendrons of different generations provides a model system for understanding the surface adhesion of molecules with multiple branches. In addition, this multiple-branch molecule may be used to mimic the sticky feet of geckos as a man-made adhesive.     

单分子水平高分子相互作用的AFM成像与单分子力谱研究

结合作者近期的研究工作,重点介绍了如何把原子力显微镜(AFM)成像及单分子力谱结合(包括原位结合或者离位结合)起来,研究高分子之间的相互作用.本文涉及生物高分子(主要是核酸-蛋白质体系)以及合成高分子体系(如聚氧乙烯,PEO)的相关研究工作.对于生物高分子体系,主要以长链核酸(如双螺旋DNA及RNA)为探针,首先利用A...     

Probability distribution analysis of force induced unzipping of DNA

We present a semimicroscopic model of dsDNA by incorporating the directional nature of hydrogen bond to describe the force induced unzipping transition. Using exact enumeration technique, we obtain the force-temperature and the force-extension curves and compare our results with the other models of dsDNA. The model proposed by us is rich enough to describe the basic mechanism of dsDNA unzipping and predicts the existence of an "eye phase." We show oscillations in the probability distribution function during unzipping. Effects of stacking energies on the melting profile have also been studied.     

Nonlinear friction characteristics between silica surfaces in high pH solution

Molecular-scale characteristics of friction forces between silica particles and silica wafers in aqueous solutions of the normal (pH 5.6) and high pH (pH 10.6) are investigated, using the lateral force measuring procedure of the atomic force microscope (AFM). Various significant differences of friction characteristics between solutions of normal and high pH's are found. In the case of solutions of normal pH, the friction force increases linearly with increasing loading force, as the Amonton's law for solid bodies indicates. However, in the case of high pH solutions, the increasing rate with the loading force is considerably reduced in the low loading region, but the value increases abruptly above a critical loading force to overcome the magnitude of friction force of normal pH above the region of very high loading. It is very interesting to know that this nonlinear force curve at high pH is independent of the atomic-scale roughness of surfaces, although the magnitude of friction is greatly influenced by the roughness in the case of normal pH. The reason why the friction at high pH is independent of the surface roughness is postulated to be due to the hairy-like layer formed on the silica surface. The existence of hairy-like layers at high pH is proven directly by the dynamic method of normal force measurements with AFM and the thickness is estimated to be at least ca. 1.3 nm.     

Study and determination of the pesticide Imidacloprid by square wave adsorptive stripping voltammetry

The kinetics of the reaction between double stranded DNA (dsDNA) and formamide is monitored at the single DNA molecule level. We find that stretching of the DNA leads to an accelerated reaction rate and to a shift in the final equilibrium concentrations. The larger the stretching force, the faster the reaction and the larger the denatured fraction of the product DNA. The single molecule kinetics is obtained from the change in the contour length of the DNA which, in turn, is measured using optical tweezers on a microbead-single DNA molecule-cover slip construct.     

The probability analysis of opening of DNA

We have studied the separation of a double stranded DNA (dsDNA), which is driven by either the temperature or force. By monitoring the probability of opening of entire base pairs along the chain, we show that the opening of a dsDNA depends not only on the sequence but also on the constraints on the chain in the experimental setups. Our results clearly demonstrate that the force-induced melting of dsDNA, whose one of the ends is constrained, is significantly different from the thermal melting, when both ends are free.     

Theory for spin-lattice relaxation of spin probes on weakly deformable DNA

The weakly bending rod (WBR) model of double-stranded DNA (dsDNA) is adapted to analyze the internal dynamics of dsDNA as observed in electron paramagnetic resonance (EPR) measurements of the spin-lattice relaxation rate, R(1e), for spin probes rigidly attached to nucleic acid-bases. The WBR theory developed in this work models dsDNA base-pairs as diffusing rigid cylindrical discs connected by bending and twisting springs whose elastic force constants are kappa and alpha, respectively. Angular correlation functions for both rotational displacement and velocity are developed in detail so as to compute values for R(1e) due to four relaxation mechanisms: the chemical shift anisotropy (CSA), the electron-nuclear dipolar (END), the spin rotation (SR), and the generalized spin diffusion (GSD) relaxation processes. Measured spin-lattice relaxation rates in dsDNA under 50 bp in length are much faster than those calculated for the same DNAs modeled as rigid rods. The simplest way to account for this difference is by allowing for internal flexibility in models of DNA. Because of this discrepancy, we derive expressions for the spectral densities due to CSA, END, and SR mechanisms directly from a weakly bending rod model for DNA. Special emphasis in this development is given to the SR mechanism because of the lack of such detail in previous treatments. The theory developed in this paper provides a framework for computing relaxation rates from the WBR model to compare with magnetic resonance relaxation data and to ascertain the twisting and bending force constants that characterize DNA.     

Loading Dynamics of a Sliding DNA Clamp

Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E. coli β clamp in real time by using single‐molecule FRET (smFRET). Three successive FRET states were retained for 0.3 s, 0.7 s, and 9 min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3 s, and after 0.7 s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9 min. An additional single‐molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single‐molecule polarization and FRET studies thus revealed the real‐time dynamics of the ATP‐hydrolysis‐dependent 3D conformational change of the β clamp during loading at a ss/dsDNA junction.     

Stepwise self-assembly of DNA tile lattices using dsDNA bridges

The simple helical motif of double-strand DNA (dsDNA) has typically been judged to be uninteresting for assembly in DNA-based nanotechnology applications. In this letter, we demonstrate construction of superstructures consisting of heterogeneous DNA motifs using dsDNA in conjunction with more complex, cross-tile building blocks. Incorporation of dsDNA bridges in stepwise assembly processes can be used for controlling length and directionality of superstructures and is analogous to the "reprogramming" of sticky-ends displayed on the DNA tiles. Two distinct self-assembled DNA lattices, fixed-size nanoarrays, and extended 2D crystals of nanotracks with nanobridges, are constructed and visualized by high-resolution, liquid-phase atomic force microscopy.     

无胶筛分芯片电泳研究贝类毒素-软骨藻酸与寡聚DNA的相互作用

近年来,我国日趋严重的赤潮已给海洋渔业带来了极大的损失,也对人类的健康构成威胁．赤潮毒素可对海洋养殖产生严重的污染,食用贝类所富集的贝类毒素严重损害食用者身体健康．根据其中毒症状,贝类毒素被分为4大类：引起腹泻性中毒(Diarrhetic Shellfish Poisoning,DSP)的贝类毒素;引起麻痹性中毒(Paralytic Shellfish Poisoning,PSP)的贝类毒素;     

Oscillatory loading of a viscoelastic adhesive contact

The cycle of loading and unloading of a spherically-tipped probe against an adhesive, viscoelastic plane specimen is studied by numerical integration of the relations between crack speed and apparent surface energy previously found for a linear 3-element viscoelastic solid with a Maugis-Dugdale law of force across the crack. It is found that even when the rate of loading is so slow that the loading and unloading curves almost coincide, suggesting purely elastic behaviour, the pull-off force can be appreciably greater than the elastic (JKR) value. When the normal force is modulated with a small amplitude sinusoidal variation during unloading--in order to find the contact stiffness--the contact radius barely changes, and the stiffness is close to that for a rigid flat punch instead of having the expected JKR value.     

Rupture force of single supramolecular bonds in associative polymers by AFM at fixed loading rates

Atomic-force-microscopy-based single-molecule force spectroscopy (AFM-SMFS) was used to study the bond strength of self-complementary hydrogen-bonded complexes based on the 2-ureido-4[1H]-pyrimidinone (UPy) quadruple H-bond motif in hexadecane (HD). The unbinding force corresponding to single UPy-UPy dimers was investigated at a fixed piezo retraction rate in the nonequilibrium loading rate regime. The rupture force of bridging supramolecular polymer chains formed between UPy-functionalized substrates and AFM tips in the presence of a bis-UPy derivative was found to decrease with increasing rupture length. The rupture length was identified as the chain length of single, associating polymers, which allowed us to determine the number of supramolecular bonds (N) at rupture. The rupture force observed as a function of N was in quantitative agreement with the theory on uncooperative bond rupture for supramolecular linkages switched in a series. Hence, the value of the dimer equilibrium constant Keq=(1.3+/-0.5) x 10(9) M(-1), which is in good agreement with previously estimated values, was obtained by SMFS of supramolecular polymers at a single loading rate.