Control of macromolecular structure and function using covalently attached double-stranded DNA constraints

The biophysical properties of DNA suggest its use for applications beyond serving as the genetic material. Several recent reports describe the use of covalently attached double-stranded DNA for controlling the structures of other macromolecules such as protein and RNA. These exploitations of DNA rigidity are conceptually distinct from many other studies in the area of "DNA nanotechnology". Double-stranded DNA constraints provide a means of introducing selective tension onto other molecules. This should facilitate fundamental investigations of macromolecular folding landscapes and tertiary interactions, as well as allow study of the mechanotransduction of biochemical signals. Use of a DNA constraint as the key element of a sensor has already been demonstrated, and such applications will be enhanced by improvements in the signal readout methods. If practical challenges such as delivery and stability can be addressed, these new efforts may also enable development of selective sensors for in vivo applications.     

Characterization of direct cellulase immobilization with superparamagnetic nanoparticles

Abstract

Methods of cellulase immobilization on magnetic particles via glutaraldehyde binding were studied. The binding was confirmed by transmission electronic microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and vibrating sample magnetometry (VSM). Samples analyzed by TEM and XRD showed that the magnetic particles with or without bound cellulase were all nanosized particles with a mean diameter of 11.5 nm, and the binding process did not cause significant changes in particle size and structure. Analysis by FTIR showed that the binding of cellulase to the magnetic nanoparticles might be via covalent bonding between residual amine groups on Fe₃O₄ nanoparticles and amine groups of the cellulase. The VSM analysis showed that magnetic nanoparticles with or without bound cellulase were all superparamagnetic. The immobilized cellulase had a wider pH and temperature range and improved storage stability compared with the free enzyme. Determination of the Michaelis constants revealed that the immobilized cellulase had a greater affinity for the cellulosic substrate than the free enzyme. The immobilized cellulase showed better performance on hydrolysis of steam-exploded corn stalks than of bleached sulfite bagasse pulp.     

DAPI derivative: a fluorescent DNA dye that can be covalently attached to biomolecules

The preparation of a DAPI (4′,6-diamidino-2-phenylindole) derivative is described. The resulting derivative retains the fluorogenic property upon binding to double-stranded DNA. Its ability for bioconjugation through amide linkage is demonstrated.     

Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles

Functionalized Fe(3)O(4) nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl chitosan nanoparticles (Fe(3)O(4) (PEG+CM-CTS)) was about 15 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. The adsorption capacity of lysozyme onto the superparamagnetic Fe(3)O(4) (PEG+CM-CTS) nanoparticles was determined by changing the medium pH, temperature, ionic strength and the concentration of lysozyme. The maximum adsorption loading reached 256.4 mg/g. Due to the small diameter, the adsorption equilibrium of lysozyme onto the nanoparticles reached very quickly within 20 min. The adsorption equilibrium of lysozyme onto the superparamagnetic nanoparticles fitted well with the Langmuir model. The nanoparticles were stable when subjected to six repeated adsorption-elution cycles. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified from chicken egg white in a single step had higher purity, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Considering that the superparamagnetic nanoparticles possess the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.     

Preparation of immobilized alpha-amylase covalently attached to granular polyacrylonitrile

In this report, alpha-Amylase originating from Bacillus subtilis (liquefying type) was immobilized on partially imidoesterized polyacrylonitrile (PAN) by covalent bonding. For the preparation of immobilized alpha-amylase, which has a high activity and high stability to repeated use, the optimum conditions for the preparation reaction were investigated. The optimum conditions for the preparation reaction were quantified on the basis of the enzymatic activity, the preservation of the activity during repeated use in batch process and the protein content on the support. Further-more, enzymatic properties of immobilized alpha-amylase prepared at optimum conditions were compared with the native enzyme. The optimum temperature and reaction time for the imidoes-terification reaction were 30 degrees c and 6 h, respectively, whereas those of the amidinatin reaction were 30-40 degrees C and more than 3 h, respectively; the optimum pH range was 9-10. Immobilized alpha-amylase prepared at the optimum conditions was very stable against the repeated use and had more than 90% of relative to activity of the first use after the tenth procedure. The initial reaction rate of immobilized alpha-amylase was lower than native alpha-amylase, but same amount of reducing sugars were produced after the reaction passed for more than 90 min. The immobilized alpha-amylase was less stabel at the high temperature and the more basic media. However, after long incubation time, immobilized alpha-amylase was more stable than the native enzyme in exposure to heat and a storng base.     

Nucleic acid hybridization using DNA covalently coupled to cellulose.

We describe a method for linking RNA and DNA covalently to finely divided cellulose through a diazotized aryl amine, which reacts primarily with guanine and uracil (thymine) residues of single strands. The high efficiency of coupling and high capacity of the cellulose for nucleic acid make possible a product with as much as 67 mug of nucleic acid per mg of cellulose. The product is especially suitable for hybridization experiments where very low backgrounds are important, and it is stable in 99% formamide at 80 degrees C so that hybridized nucleic acid can be recovered easily. Full length linear Simian Virus 40 (SV40) DNA, produced by cleavage of SV40(I) DNA with S1 nuclease, can be coupled to diazo cellulose with an efficiency of 80-90%, and is effective in hybridization experiments with SV40 DNA, complementary RNA synthesized in vitro from SV40(I) DNA with E. coli RNA polymerase, and the SV40-specific fraction of total RNA from SV40-infected and transformed cells. In these experiments an excess of cellulose-bound DNA was used, and the efficiency of hybridization was about 90% when ribonuclease treatment of the hybrids was omitted.     

Electrical detection of the temperature induced melting transition of a DNA hairpin covalently attached to gold interdigitated microelectrodes

The temperature induced melting transition of a self-complementary DNA strand covalently attached at the 5′ end to the surface of a gold interdigitated microelectrode (GIME) was monitored in a novel, label-free, manner. The structural state of the hairpin was assessed by measuring four different electronic properties of the GIME (capacitance, impedance, dissipation factor and phase angle) as a function of temperature from 25°C to 80°C. Consistent changes in all four electronic properties of the GIME were observed over this temperature range, and attributed to the transition of the attached single-stranded DNA (ssDNA) from an intramolecular, folded hairpin structure to a melted ssDNA. The melting curve of the self-complementary single strand was also measured in solution using differential scanning calorimetry (DSC) and UV absorbance spectroscopy. Temperature dependent electronic measurements on the surface and absorbance versus temperature values measured in solution experiments were analyzed assuming a two-state process. The model analysis provided estimates of the thermodynamic transition parameters of the hairpin on the surface. Two-state analyses of optical melting data and DSC measurements provided evaluations of the thermodynamic transition parameters of the hairpin in solution. Comparison of surface and solution measurements provided quantitative evaluation of the effect of the surface on the thermodynamics of the melting transition of the DNA hairpin.     

Application of superparamagnetic nanoparticles in purification of plasmid DNA from bacterial cells

The aim of this study was to develop a simple and rapid method for purification of ultrapure supercoiled plasmid DNA with high yields from bacterial cultures. Nanosized superparamagnetic nanoparticles (Fe3O4) were prepared by chemical precipitation method using Fe2+, Fe3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe3O4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The nanoparticles were characterized by transmission electron microscopy, X-ray diffraction, Fourier transformation infrared spectroscopy and superconducting quantum interference device magnetometer. The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 35 microg of high-purity (A260/A280 ratio=1.87) plasmid DNA was isolated from 3ml of overnight bacterial culture. EGFP expression was detected by fluorescent microscopy in the transformed E. coli cells, indicating the biological activities of DNA fragments were retained after purified from magnetic nanobeads. The protocol, starting from the preparation of bacterial lysate and ending with purified plasmids takes less than 10 min. Thus, the separation and purification qualities of PEI-modified magnetic nanobeads as well as its ease of use surpass those of conventional anion-exchange resins.     

Assembly system of direct modified superparamagnetic iron oxide nanoparticles for target-specific MRI contrast agents

We report the direct modification of SPIOs with a biomolecule and the target-specific assembly system of these modified SPIOs for using MRI contrast agents. The transverse relaxation rate of the aqueous solutions containing the modified SPIOs was altered by the dispersion state.     

Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids

As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9.     

A glycophospholipid covalently attached to the C-terminus of the Thy-1 glycoprotein

The Thy-1 glycoprotein is a very abundant cell surface molecule of rat thymocytes and neuronal cells with the properties of a molecule that inserts into the lipid bilayer. The hydrophobicity is due to a glycophospholipid component covalently attached to the carboxy group of the C-terminal cysteine residue. The mature glycoprotein does not contain a stretch of hydrophobic amino acids that could traverse the membrane bilayer. These findings present a new mode of membrane attachment for a cell surface molecule that can mediate lymphokine release and cell division after cross-linking by antibodies.     

Fluorescent DNA nanotags featuring covalently attached intercalating dyes: synthesis, antibody conjugation, and intracellular imaging

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.     

Repair of DNA covalently linked to protein

A potentially lethal form of DNA/RNA modification, a cleavage complex, occurs when a nucleic acid-processing enzyme that acts via a transient covalent intermediate becomes trapped at its site of action. A number of overlapping pathways act to repair these lesions and many of the enzymes involved are those that catalyze recombinational-repair processes. A protein, Tdp1, has been identified that reverses cleavage-complex formation by specifically hydrolyzing a tyrosyl-DNA phosphodiester bond. The study of these pathways is both interesting and pertinent as they modulate the effectiveness of many antitumor/antibacterial drugs that act by stabilizing cleavage-complexes in vivo.     

Quantitative analysis of specific labelled RNA''S using DNA covalently linked to diazobenzyloxymethyl-paper.

Substantial amounts of DNA (at least 25 microgram per cm2) can be stably bound to diazobenzyloxymethyl (DBM)-paper. Complementary RNA will hybridize to the DNA paper almost completely in 24 hours. Using several different conditions of hybridization and washing, the background of RNA bound non-specifically is very low (between 0.01 and 0.02%) and the efficiency of hybridization is very high (75 to 50% of complementary RNA is bound and retained through the washing procedure). Because the DNA is bound to the paper convalently, it is retained through all the washing and elution steps, and the DNA papers can be re-used many times.     

Inactivation of Pseudomonas aeruginosa PA01 biofilms by hyperthermia using superparamagnetic nanoparticles

The primary goal of this study was to develop a new strategy to inactivate bacterial biofilms using the thermal stress derived from superparamagnetic iron oxide nanoparticles (SPIONs) in an alternating current (AC) magnetic field. A large number of studies have examined the inactivation of bacterial biofilms using antimicrobial agents; however, there have been no attempts to inactivate biofilms by hyperthermia using SPIONs. In this study, a SPION solution was added to Pseudomonas aeruginosa (P. aeruginosa) PA01 biofilm, and heat was generated by placing the nanoparticle-containing biofilm in an AC magnetic field. The heating temperature was dependent on the concentration of the added SPION solution. More than 4 log inactivation of the PA01 biofilm was obtained using a 60 mg mL⁻¹ SPION solution in 8 min, and this resulted in a dramatic disintegration of the bacterial cell membrane in the biofilm. This inactivation was largely due to the thermal effect. Local heating of a specific area is also possible using this method, and the heating temperature can be easily adjusted by controlling the concentration of the SPION solution. Therefore, hyperthermia using magnetic nanoparticles holds promise as an effective tool for inactivating the bacterial biofilm.     

Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA

A simple, high-throughput, low-cost polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) protocol that detects the presence of 4 fg of DNA from four bacterial fish pathogens Yersinia ruckeri, Tenacibaculum maritimum (formerly Flexibacter maritimus), Lactococcus garvieae and Aeromonas salmonicida was developed. DNA amplification was undertaken in a biphasic system with free and bound PCR that are achieved in the one NucleoLink tube. Solid-phase amplicons were detected using biotin labelled hybridization probes and visualised colourimetrically with streptavidin-alkaline phosphatase and p-nitrophenylphosphate as substrate. PCR and hybridization took less than 8 h to perform with maximum signal output for femtogram amounts of template DNA achieved within 24 h. Implementation and optimization of the protocol is discussed.     

Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The Schiff base intermediate formed during this reaction between Escherichia coli Fpg and DNA was trapped by reduction with sodium borohydride, and the structure of the resulting covalently cross-linked complex was determined at a 2.1-A resolution. Fpg is a bilobal protein with a wide, positively charged DNA-binding groove. It possesses a conserved zinc finger and a helix-two turn-helix motif that participate in DNA binding. The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA, which is sharply kinked at the lesion site. Residues Met-73, Arg-109, and Phe-110 are inserted into the DNA helix, filling the void created by nucleotide eversion. A deep hydrophobic pocket in the active site is positioned to accommodate an everted base. Structural analysis of the Fpg-DNA complex reveals essential features of damage recognition and the catalytic mechanism of Fpg.     

Coupled oxidation of heme covalently attached to cytochrome b562 yields a novel biliprotein

A variant of Escherichia coli cytochrome b(562) with covalently attached heme can be converted to a biliverdin-containing protein in two distinct stages by coupled oxidation and acid hydrolysis. The first stage of coupled oxidation yields a stable verdoheme-containing protein. This verdoheme protein is unusual in three respects. First, the verdoheme group is covalently bound to the protein through a c-type thioether linkage. Second, the oxidation stops at the verdoheme stage, and finally, this is the first report of verdoheme generated from a heme protein with exclusive methionine ligation to the heme iron. In addition, the oxidation process does not require denaturation of the protein. The product has been characterized by optical spectroscopy, ESI mass spectrometry, and (1)H NMR. The NMR data show that the predominant product is the result of oxidation at the alpha-meso carbon. A collective evaluation of data on the topic suggests that the electronic structure of the heme, not protein steric effects, is the main factor in controlling the regiospecificity of the oxidation site. In the second stage of conversion to a biliprotein, we demonstrate that the verdoheme ring can be opened by treatment with aqueous formic acid to give alpha-biliverdin covalently attached to the folded protein. This product, a protein-bound linear tetrapyrrole as characterized by optical spectroscopy and mass spectrometry, is an example of a phycobilin chromophore that has not been observed previously.