DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.     

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.     

Cell microarrays: an emerging technology for the characterization of antibodies

The possibility to miniaturize and parallelize biological assays has a great impact on the development of biomedical technologies. Here, we describe a simple, miniaturized, and parallelized method employing entire cells from different cell lines displaying a protein of interest on their surface, which were immobilized on a microarray slide. Antibodies were added to these cellular microarrays, and their specific binding to the cell surface proteins was monitored using appropriate fluorescently labeled detection molecules. This new method is applicable for rapidly screening cell surface-specific antibodies with respect to selectivity and cross-reactivity.     

凝胶基片的制备与应用研究

在Bind-Silane^R处理的玻片上交联聚丙烯酰胺凝胶层(15mm×15mm×20μm),戊二醛活化。与末端氨基修饰的寡核苷酸片段共价结合制成芯片。这种芯片能够区分液相中序列不同的Cy3标记的目标核酸。与平面基片相比,凝胶基片具有背景低、固定探针量高、杂交时间短的优点。将细胞因子IL-4、IL-5、IL-6、IL-7、ANG、I-309和VEGF的单克隆抗体加样于凝胶基片上制成蛋白质芯片,对乳腺癌患者和正常人的血清进行检测,发现乳腺癌患者细胞因子IL-4、IL-5、I-309和VEGF的表达量高于正常人的表达量,对临床诊断具有重要的参考意义。     

Protein microarrays: Reduced autofluorescence and improved LOD

In protein microarray performance, the choice of an appropriate surface is a crucial factor. Three‐dimensional substrates like nitrocellulose are known to have higher binding capacities than planar surfaces. Furthermore, they can enable the immobilization of proteins in a functional manner. One disadvantage of today's nitrocellulose‐based microarrays is the high background fluorescence, which can interfere with the detection of low‐abundance proteins. We have developed an innovative black nitrocellulose membrane‐based protein microarray that exhibits low autofluorescence in combination with increased sensitivity and improved LOD (limit of detection). The applicability of the novel material was demonstrated with main focus on reversed‐phase microarray experiments. In comparison to various commercially available microarrays, a higher sensitivity in regard to the spotted protein was achieved. In contrast to other porous nitrocellulose‐based microarrays, the black nitrocellulose provides a significant lower autofluorescence and background intensity.     

Fluorescence resonance energy transfer between bovine serum albumin and fluoresceinamine

Physical binding‐mediated organic dye direct‐labelling of proteins could be a promising technology for bio‐nanomedical applications. Upon binding, it was found that fluorescence resonance energy transfer (FRET) occurred between donor bovine serum albumin (BSA; an amphiphilic protein) and acceptor fluoresceinamine (FA; a hydrophobic fluorophore), which could explain fluorescence quenching found for BSA. FRET efficiency and the distance between FA and BSA tryptophan residues were determined to 17% and 2.29 nm, respectively. Using a spectroscopic superimposition method, the saturated number of FAs that bound to BSA was determined as eight to give a complex formula of FA₈–BSA. Finally, molecular docking between BSA and FA was conducted, and conformational change that occurred in BSA upon binding to FA molecules was also studied by three‐dimensional fluorescence microscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

Design and synthesis of the BODIPY–BSA complex for biological applications

A quinoxaline‐functionalized styryl–BODIPY derivative (S1) was synthesized by microwave‐assisted Knoevenagel condensation. It exhibited fluorescence enhancement upon micro‐encapsulation into the hydrophobic cavity of bovine serum albumin (BSA). The S1–BSA complex was characterized systematically using ultraviolet (UV)–visible absorption, fluorescence emission, kinetics, circular dichroism and time‐resolved lifetime measurements. The binding nature of BSA towards S1 was strong, and was found to be stable over a period of days. The studies showed that the S1–BSA complex could be used as a new biomaterial for fluorescence‐based high‐throughput assay for kinase enzymes.     

Spectroscopic analysis on structure–affinity relationship in the interactions of different oleanane‐type triterpenoids with bovine serum albumin

Oleanane‐type triterpenoids serve as an important group of plant secondary metabolites with a variety of biological activities and the C‐3 position substitution pattern is a significant structural feature for their biological activities. Three selected oleanane‐type triterpenoids (glycyrrhizin, glycyrrhetinic acid, and carbenoxolone) bearing different substituents (glucuronic acid dimer, hydroxyl, and succinyl groups) at the C‐3 position were studied for their affinities to bind bovine serum albumin (BSA) by steady‐state fluorescence, synchronous, three‐dimensional fluorescence and ultraviolet–visible (UV–vis) absorption spectra. The binding mechanism of the triterpenoids to BSA is due to the formation of the triterpenoids–BSA complex and the binding affinity is strongest for carbenoxolone and ranked in the order carbenoxolone > glycyrrhetinic acid > glycyrrhizin. The thermodynamic parameters calculated at different temperatures showed that triterpenoids binding to BSA primarily depended on hydrophobic interaction and hydrogen bonding. The distance between the bound triterpenoid and BSA was determined on the basis of the Förster's energy transfer theory. Displacement experiments using phenylbutazone and ibuprofen showed the binding site of triterpenoids on BSA at subdomain IIA (Sudlow's site I). The effect of triterpenoids on BSA conformation was analyzed by UV–vis absorption, and synchronous and three‐dimensional fluorescence spectra. These results revealed that the C‐3 position substitution pattern significantly affects the structure–affinity relationships of oleanane‐type triterpenoid binding to BSA and further affects the bioavailability of triterpenoids in the blood circulatory system. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparative studies on the interactions of dihydroartemisinin and artemisinin with bovine serum albumin using spectroscopic methods

The interactions of dihydroartemisinin (DHA) and artemisinin (ART) with bovine serum albumin (BSA) have been investigated using fluorescence, UV/vis absorption and Fourier transform infrared (FTIR) spectra under simulated physiological conditions. The binding characteristics of DHA/ART and BSA were determined by fluorescence emission and resonance light scattering (RLS) spectra. The quenching mechanism between BSA and DHA/ART is static. The binding constants and binding sites of DHA/ART–BSA systems were calculated at different temperatures (293, 298, 304 and 310 K). According to Förster non‐radiative energy transfer theory, the binding distance of BSA to DHA/ART was calculated to be 1.54/1.65 nm. The effect of DHA/ART on the secondary structure of BSA was analyzed using UV/vis absorption, FTIR, synchronous fluorescence and 3D fluorescence spectra. In addition, the effects of common ions on the binding constants of BSA–DHA and BSA–ART systems were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Thermodynamic dissection of the polymerizing and editing modes of a DNA polymerase

DNA polymerases with intrinsic proofreading activity interact with DNA primer/templates in two distinct modes, corresponding to the complexes formed during the 5'-3' polymerization or 3'-5' editing of a nascent DNA chain. Thermodynamic measurements designed to quantify the energetic contributions of individual DNA-protein contacts in either the polymerizing or editing complexes are complicated by the fact that both species exist in solution and are not resolved in conventional DNA-protein binding assays. To overcome this problem, we have developed a new binding analysis that combines information from steady-state and time-resolved fluorescence experiments and uses the Klenow fragment of Escherichia coli DNA polymerase I (KF) and fluorescently labeled primer/template oligonucleotides as a model polymerase-DNA system. Steady-state fluorescence titrations are used to evaluate the overall affinity of KF for the primer/template, while time-resolved fluorescence anisotropy is used to quantify the equilibrium fractions of the primer/template bound in the polymerizing and editing modes. From a combined analysis of both data, the equilibrium constant and hence standard free energy change associated with each binding mode can be obtained unequivocally. This method is initially used to determine the equilibrium constants describing binding of a correctly base-paired primer/template to the 5'-3' polymerase and 3'-5' exonuclease sites of KF. It is then extended to quantify the extent to which these parameters are affected by the introduction of mismatches into the primer/template, and by rearrangement of specific side-chains in the exonuclease domain of the protein. While these perturbants were originally designed to demonstrate the utility of our new approach, they are also relevant in their own right since they have helped identify some hitherto unknown determinants of polymerase fidelity.     

Effect of triazole‐tryptophan hybrid on the conformation stability of bovine serum albumin

The effect of a potent antimicrobial compound bearing 1,2,3‐triazole core and a tryptophan tail, triazole‐tryptophan hybrid (TTH), with bovine serum albumin (BSA) have been explored using various spectroscopic and molecular docking methods. Studies revealed that TTH strongly quenches the intrinsic fluorophore of BSA by a static quenching mechanism. Time‐resolved fluorescence spectra further confirmed the involvement of static quenching for TTH–BSA system. The calculated thermodynamic parameters; ΔH, ΔS, and ΔG showed that the binding process was spontaneous, exothermic and entropy driven. Synchronous fluorescence, three‐dimensional (3D) fluorescence and circular dichroism data revealed that TTH induces the structural alteration in BSA and enhances its stability. In silico study of TTH–BSA system showed that it binds with BSA at the site I of subdomain IIA. Both the experimental and in silico study showed that the hydrophobic and electrostatic interactions play a major role in TTH–BSA binding.     

Characterization of domain-specific interaction of synthesized dye with serum proteins by spectroscopic and docking approaches along with determination of in vitro cytotoxicity and antiviral activity

The interaction between a synthesized dye with proteins, bovine, and human serum albumin (BSA, HSA, respectively) under physiological conditions has been characterized in detail, by means of steady-state and time-resolved fluorescence, UV–vis absorption, and circular dichroism (CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to divulge the actual quenching mechanism. From the thermodynamic observations, it is clear that the binding process is a spontaneous molecular interaction, in which van der Waals and hydrogen bonding interactions play the major roles. The UV–vis absorption and CD results confirm that the dye can induce conformational and micro-environmental changes of both the proteins. In addition, the dye binding provokes the functionality of the native proteins in terms of esterase-like activity. The average binding distance (r) between proteins and dye has been calculated using FRET. Cytotoxicity and antiviral effects of the dye have been found using Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking simulation reveals the probable binding location of dye within the sub-domain IIA of HSA and IB of BSA.     

A fluorescence study of differently substituted 3‐styrylindoles and their interaction with bovine serum albumin

Interaction of 3‐styrylindoles 1–8 viz. 3‐(2‐phenylethenyl‐E)‐NH‐indole (1), 3‐[2‐(4‐nitrophenyl)ethenyl‐E]‐NH‐indole (2), 5‐bromo‐3‐[2‐(4‐nitrophenyl)ethenyl‐E]‐NH‐indole (3), 5‐methoxy‐3‐[2‐(4‐nitrophenyl)ethenyl‐E]‐NH‐indole (4), 3‐[2‐(4‐cyanophenyl)ethenyl‐E]‐NH‐indole (5), 3‐[2‐(4‐cyanophenyl)ethenyl‐E]‐N‐ethylindole (6), 5‐bromo‐3‐[2‐(4‐chlorophenyl)ethenyl‐E]‐NH‐indole (7) and 5‐methoxy‐3‐[2‐(4‐chlorophenyl)ethenyl‐E]‐NH‐indole (8) with bovine serum albumin (BSA) was examined by UV–vis and steady‐state fluorescence spectroscopy. The fluorescence intensity of 1–8 increases with the increasing BSA concentration. Upon binding with BSA, while 1 and 5–8 show a blue shift in their λ_{f max}, 2–4 do not exhibit such behavior. Compounds 1–8 also quench the 345 nm fluorescence of BSA in phosphate buffer (λ_ex, 280 nm). These compounds intercalate in the hydrophobic regions of BSA, as evidenced by the determination of BSA binding site micropolarity using compounds 2–8. As evidenced by the estimation of energy transfer efficiency and distance between the donor (BSA‐Trp‐212) and the acceptor (3‐styrylindoles), the halo‐substituted compounds 3 and 7 interact with BSA more effectively than the other 3‐strylindoles. These compounds have potential for use as neutral and hydrophobic fluorescence probes for examining the microenvironments in proteins, polymers, micelles, etc. Copyright © 2008 John Wiley & Sons, Ltd.     

The prevention of adsorption of interferents to radiolabelled protein by Tween 20

Radiolabels are often used to quantitatively determine the amount of protein immobilized on chromatographic supports, immunochemical plates and biosensor surfaces. Bovine serum albumin (BSA) was chosen as a model protein for quantitative deposition studies. BSA was radioiodinated (125I-) or fluorescently labelled (fluorescein), then incubated with the following surfaces: quartz, quartz derivatized by 3-aminopropyltriethoxysilane (Qz-APTES), and Qz-APTES reacted with glutaraldehyde or tresyl chloride. The amounts of BSA immobilized to the different surfaces were compared using data from radioactivity and fluorescence assays. Irreproducible results were obtained with radioiodinated BSA due to adsorption/desorption behaviour of an unidentified radioactive species. When the non-ionic detergent Tween 20 was added to the protein/surface incubation mixture, radiolabelled BSA gave reproducible protein binding results which agreed with fluorescent protein binding patterns. The effect of Tween 20 was due to either the binding to BSA displacing the interferent and/or the solubilization of the interferent.     

Spectroscopic and molecular modelling analysis of the interaction between ethane‐1,2‐diyl bis(N,N‐dimethyl‐N‐hexadecylammoniumacetoxy)dichloride and bovine serum albumin

Several spectroscopic approaches namely fluorescence, time‐resolved fluorescence, UV‐visible, and Fourier transform infra‐red (FT‐IR) spectroscopy were employed to examine the interaction between ethane‐1,2‐diyl bis(N,N‐dimethyl‐N‐hexadecylammoniumacetoxy)dichloride (16‐E2‐16) and bovine serum albumin (BSA). Fluorescence studies revealed that 16‐E2‐16 quenched the BSA fluorescence through a static quenching mechanism, which was further confirmed by UV–visible and time‐resolved fluorescence spectroscopy. In addition, the binding constant and the number of binding sites were also calculated. The thermodynamic parameters at different temperatures (298 K, 303 K, 308 K and 313 K) indicated that 16‐E2‐16 binding to BSA is entropy driven and that the major driving forces are electrostatic interactions. Decrease of the α‐helix from 53.90 to 46.20% with an increase in random structure from 22.56 to 30.61% were also observed by FT‐IR. Furthermore, the molecular docking results revealed that 16‐E2‐16 binds predominantly by electrostatic and hydrophobic forces to some residues in the BSA sub‐domains IIA and IIIA. Copyright © 2015 John Wiley & Sons, Ltd.     

Explication of bovine serum albumin binding with naphthyl hydroxamic acids using a multispectroscopic and molecular docking approach along with its antioxidant activity

In the present investigation, the protein‐binding properties of naphthyl‐based hydroxamic acids (HAs), N‐1‐naphthyllaurohydroxamic acid ( 1 ) and N‐1‐naphthyl‐p‐methylbenzohydroxamic acid ( 2 ) were studied using bovine serum albumin (BSA) and UV–visible spectroscopy, fluorescence spectroscopy, diffuse reflectance spectroscopy–Fourier transform infrared (DRS–FTIR), circular dichroism (CD), and cyclic voltammetry along with computational approaches, i.e. molecular docking. Alteration in the antioxidant activities of compound 1 and compound 2 during interaction with BSA was also studied. From the fluorescence studies, thermodynamic parameters such as Gibb's free energy (ΔG), entropy change (ΔS) and enthalpy change (ΔH) were calculated at five different temperatures (viz., 298, 303, 308, 313 or 318 K) for the HAs–BSA interaction. The results suggested that the binding process was enthalpy driven with dominating hydrogen bonds and van der Waals’ interactions for both compounds. Warfarin (WF) and ibuprofen (IB) were used for competitive site‐specific marker binding interaction and revealed that compound 1 and compound 2 were located in subdomain IIA (Sudlow's site I) on the BSA molecule. Conclusions based on above‐applied techniques signify that various non‐covalent forces were involved during the HAs–BSA interaction. Therefore the resulted HAs–BSA interaction manifested its effect in transportation, distribution and metabolism for the drug in the blood circulation system, therefore establishing HAs as a drug‐like molecule.     

利用糖芯片技术检测氨基糖苷类抗生素与RNAs和蛋白质之间的相互作用

氨基糖苷类抗生素是一类广谱型抗细菌感染药物,其不断增加的细菌耐药性很大程度上限制了它的临床应用,研究和开发新型氨基糖苷类抗生素具有重要意义。将氨基糖苷类抗生素固定到玻璃片基上,制成糖芯片,再分别与荧光标记的RNAs和蛋白质杂交,通过分析杂交后的荧光信号强度检测它们之间的相互作用。结果显示,氨基糖苷类抗生素芯片可以特异性地与r RNA的A位点模拟物、I型核酶和蛋白酶结合。因此糖芯片技术不仅可以检测氨基糖苷类抗生素与r RNAs的特异性结合,而且可以应用于寻找新型RNA结合配体的研究,为快速鉴定和筛选可紧密结合RNA靶标且毒性较低的新型氨基糖苷类抗生素奠定了一定的基础。     

Investigations on the interactions of DiAmsar with serum albumins: Insights from spectroscopic and molecular docking techniques

Diamine‐sarcophagine (DiAmsar) binding to human serum albumin (HSA) and bovine serum albumin (BSA) was investigated under simulative physiological conditions. Fluorescence spectra in combination with Fourier transform infrared (FT‐IR), UV‐visible (UV–vis) spectroscopy, cyclic voltammetry (CV), and molecular docking method were used in the present work. Experimental results revealed that DiAmsar had an ability to quench the HSA and BSA intrinsic fluorescence through a static quenching mechanism. The Stern–Volmer quenching rate constant (K_sv) was calculated as 0.372 × 10³ M^‐1 and 0.640 × 10³ M^‐1 for HSA and BSA, respectively. Moreover, binding constants (K_a), number of binding sites (n) at different temperatures, binding distance (r), and thermodynamic parameters (?H°, ?S°, and ?G°) between DiAmsar and HSA (or BSA) were calculated. DiAmsar exhibited good binding propensity to HSA and BSA with relatively high binding constant values. The positive ?H° and ?S° values indicated that the hydrophobic interaction is main force in the binding of the DiAmsar to HSA (or BSA). Furthermore, molecular docking results revealed the possible binding site and the microenvironment around the bond. Copyright © 2014 John Wiley & Sons, Ltd.