Online Capillary Electrophoresis Reaction for Interaction Study of Amino Acid Modified Peptide Nucleic Acid and Proteins

Peptide nucleic acid (PNA) is a nucleic acid analog which consists of purines, pyrimidines bases, and a neutrally charged peptide backbone. The PNA has the potential as a very useful biological probe for protein analysis since it has more in vivo biological stability as compared to DNA- or RNA-based aptamers. Usually, the addition of amino acids or peptide to the PNA backbone is used to improve its water-solubility and cell-permeability, but these modifications may affect the interaction between PNA and proteins. To date, the investigation of the interaction between PNA and proteins is rare, and there is no reported study about the effects of modifications. In this work, we designed two types of amino acid modified PNAs, (Lys)₂-PNA and (Glu)₂-PNA, which kept the same base sequence with 15-mer thrombin aptamer and had two basic lysine and two acidic glutamic acid residues on N-terminal of the peptide backbone, respectively. To rapidly assess the binding affinity and specificity of modified PNA and proteins, the online CE reaction method was developed to analyze the interactions of (Lys)₂-PNA/(Glu)₂-PNA and three proteins: thrombin (THB), single-strand DNA-binding protein (SSB) and human serum albumin (HSA). Meanwhile, the interactions of (Lys)₂-PNA/(Glu)₂-PNA and thrombin were compared with that of the corresponding complementary base sequence (Lys)₂-cPNA/(Glu)₂-cPNA and thrombin. The online CE reaction results showed that the interaction of (Lys)₂-PNA and (Glu)₂-PNA with three proteins was in the order of THB > SSB > HSA. However, (Lys)₂-PNA and (Lys)₂-cPNA showed similar binding affinity with thrombin; while the binding affinity of (Glu)₂-PNA with thrombin was stronger than that of (Glu)₂-cPNA with thrombin. Moreover, the binding constant K_b of (Glu)₂-PNA and three proteins was determined by affinity capillary electrophoresis (ACE). The online CE reaction eliminates the requirement of incubation, and thus it is fast in detection, and easy to operate with minimum cost. The method is particularly suitable for the interaction studies of expensive modified PNAs and proteins, and can assist the design of PNA probe that binds to proteins.     

The importance of inter-molecular forces in crystal structure analysis: Solid state study of the diag-to-lat isomerization of (η5-C5H4Me)Re(CO)2Br2

Single crystal X-ray structures of diag and lat-(⁵-C₅H₄Me)Re(CO)₂Br₂ have been determined. The diag form crystallizes in the triclinic space group , a = 6.751(2), b = 8.537(1), c = 9.758(1) Å = 96.70(1), = 93.15(2), = 104.96(2)°, V = 534.8(2) ų, Z = 2. The lat form is monoclinic, P2₁/c, a = 11.820(1), b = 7.133(1), c = 12.924(1) Å = 98.278(8)°, V = 1078.2(2) ų, Z = 4. The unit cell volumes (per Z) of the two isomers differ by ca 1%. Molecular modelling reveals an energy difference between the two isomers of 1 kJ mol^–1 or less. The XRD powder diffraction patterns for the lat isomer produced by crystallization from solution or prepared by thermal isomerization are identical. The evidence thus suggests that the solid state isomerization reaction is a novel example of a reaction which yields a molecular structure determined by crystal packing forces.     

Mathematical modeling of polymer-induced flocculation by charge neutralization

A detailed mathematical model for flocculation of colloidal suspensions in presence of salts and polymers is described and validated. In former case, the classical DLVO theory, which accounts for relevant variables such as pH and salt concentration, is incorporated into a geometrically sectioned discrete population balance model. For processes involving polymers, flocculation via simple charge neutralization is modeled using a modified DLVO theory in which the effect of adsorbed polymer layers on van der Waals attraction is included. The fractal dimension of aggregates is obtained by dynamic scaling of experimental data for time evolution of mean aggregate size. The particle surface potential is assumed to be approximately equal to the zeta potential. The model predictions are in close agreement with experimental results for flocculation of colloidal hematite suspensions in the presence of KCl and polyacrylic acid at different concentrations. In particular, given values of model parameters, e.g., Hamaker constant, fractal dimension, surface potential, and thickness of adsorbed polymer layer, the model can realistically describe the kinetics of flocculation by a simple charge neutralization mechanism and track the evolution of floc size distribution. Representative examples of sensitivity of the flocculation model to perturbations in surface potential and fractal dimension and to modification in the DLVO theory for polymer-coated particles are included.     

In‐capillary detection of fast antibody‐peptide binding using fluorescence coupled capillary electrophoresis

Herein, we report a technique for detecting the fast binding of antibody‐peptide inside a capillary. Anti‐HA was mixed and interacted with FAM‐labeled HA tag (FAM‐E₄) inside the capillary. Fluorescence coupled capillary electrophoresis (CE‐FL) was employed to measure and record the binding process. The efficiency of the antibody‐peptide binding on in‐capillary assays was found to be affected by the molar ratio. Furthermore, the stability of anti‐HA‐FAM‐E₄ complex was investigated as well. The results indicated that E₄YPYDVPDYA (E₄) or TAMRA‐E₄YPYDVPDYA (TAMRA‐E₄) had the same binding priorities with anti‐HA. The addition of excess E₄ or TAMRA‐E₄ could lead to partial dissociation of the complex and take a two‐step mechanism including dissociation and association. This method can be applied to detect a wide range of biomolecular interactions.     

Structure and interaction of adsorbing symmetrical triblock polyampholyte solution between two planes

In this paper, the continuum self-consistent field theory(SCFT) is applied to study the structure and the interaction of the adsorption of symmetrical ABA polyampholytes(PAs) between two neutral planes. It is found that the amounts of all the conformations decrease with the increase of the charge fraction of polymer chain, and increase with the increase of the bulk salt concentration and become saturated at high bulk salt concentration. The effective interaction between the two planes presented a long-range repulsion. Splitting it into various components and relating with the dependence of the variations of the conformations on environment parameters, we try to find the origin of the total long-range interaction between the two planes.     

聚合物与表面活性剂相互作用的分子模拟研究进展

聚合物与表面活性剂复配体系已广泛应用于医药、生物、石油石化等领域。从微观上认识其相互作用机理对指导其生产实际有着重要作用,因而此方面的研究倍受关注。随着分子模拟技术的发展,聚合物与表面活性剂在分子水平上的相互作用机理研究已经被广泛开展,并获得了大量有用的信息。本文综述了耗散粒子动力学(DPD)和粗粒度分子动力学(CG-MD)在聚合物与表面活性剂相互作用方面的应用,分别对中性聚合物与离子型表面活性剂,以及带相反电荷的聚电解质和表面活性剂在溶液相和界面相的相互作用进行了阐述,并揭示了聚合物/表面活性剂聚集体结构形态的变化规律。     

铜与烟酸和8-羟基喹啉配合物的电化学行为及与DNA的相互作用

采用循环伏安法研究了铜与烟酸和8-羟基喹啉配合物(Cu(Hq)(NA)0.5(Hq=8-羟基喹啉,NA=烟酸))的电化学行为。此外,以鲱鱼精DNA为靶点,采用紫外吸收光谱、DNA粘度滴定和差分脉冲伏安法,从分子水平研究了该配合物与DNA的键合方式。结果发现:配合物的中心Cu~(2+)在循环伏安图上呈现明显的氧化还原峰,峰电流随扫描速度的增加呈增加趋势,并且与扫描速度成正比。配合物的吸收峰强度随着DNA的加入减色显著,氧化还原峰电流也随之减小,式量电位发生正移;粘度实验结果发现DNA的相对比粘度随配合物的加入而增大。这些结果表明配合物与DNA通过嵌插方式发生作用。     

Exploring the interaction of calycosin with cyclin D1 protein as a regulator of cell cycle progression in lung cancer cells

Cyclin D1 has been shown to play a pivotal role in the proliferation of lung cancer cells through regulation of cell cycle progression. Therefore, targeting this protein can be used as a potential strategy in lung cancer treatment. Calycosin has been reported to show potential anticancer effects, however, its possible anticancer mechanisms remain unclear. Therefore, in this study we aimed to explore the interaction of cyclin D1 and calycosin to determine the binding properties and probable structural changes of cyclin D1. We carried out in-depth experimental and computational binding assays of calycosin with cyclin D1 under simulated physiological environment, using intrinsic, extrinsic, synchronous fluorescence, circular dichroism, and differential scanning calorimetry (DSC) analysis. The results showed a spontaneous static mechanism driven from hydrogen bonding and van der Waals forces between hydrophilic residues of cyclin D1 with hydroxyl groups of calycosin. We determined that calycosin led to secondary and tertiary structural changes of cyclin D1 through exposure of hydrophobic residues. Also, it was determined that calycosin resulted in an apparent decrease in the heat capacity changes (ΔCp) and midpoint of unfolding transition (Tm) values of cyclin D1. Cellular studies also indicated that calycosin caused the inhibition of lung cancer cell proliferation through cell cycle arrest at G1 phase, which may be due to denaturation of cyclin D1, although it needs further investigation in the future studies. In general, this study may provide useful preliminary data about the development of calycosin-based anticancer platforms.     

The adsorption of dopamine on gold and its interactions with iron(III) ions studied by microcantilevers

The adsorption of dopamine (DA) molecules on gold and their interactions with Fe³⁺ were studied by a microcantilever in a flow cell. The microcantilever bent toward the Au side with the adsorption of DA due to the change of surface stress induced by the intermolecular hydrogen bonds of DA or the charge transfer effect between adsorbates and the substrate. The interaction process between DA adsorbates and Fe³⁺ was revealed by the deflection curves of microcantilever. As indicated by the appearance of a variation during the decline of curves, two steps were observed in the curve at relative high concentrations of Fe³⁺. In this case, Fe³⁺ reacted with DA molecules only in the outer layers and the complexes removed with solution. Then Fe³⁺ reacted further with DA molecules forming the surface complex in the first layer next to the gold. At this stage, the stability of surface complexes was time dependent, i.e., unstable initially and stable finally. This may be due to the surface complexes change from mono-dentate to bi-dentate complexes. In another case, i.e., at relative low concentration of Fe³⁺, only the first step was observed as indicated by the absence of a variation. X-ray photoelectron spectroscopy (XPS) and cycling voltammetry (CV) results provided complementary evidence for the result of microcantilever and proposal. As low as 5 × 10⁻¹⁰ M Fe³⁺ was detected by DA modified microcantilever with a good selectivity over other common metal ions.