Gold nanoparticles used as a carrier enhance production of anti-hapten IgG in rabbit: a study with azobenzene-dye as a hapten presented on the entire surface of gold nanoparticles

The azobenzene moiety, well-known not only for its reversible cis-to-trans photoisomerization but also as a hapten, does not induce antibodies on its own, but it reacts with antibodies raised against conjugates with protein carriers. Hence we selected azobenzene dye as an indicator to assess the possibility of having gold nano-particles act as an immunological carrier instead of protein carriers. In rabbits, we confirmed an in vivo response against azobenzene dye presented on the entire surface of gold nanoparticles (azo-nanoparticles), where the gold nanoparticles appeared to play a role as a carrier for the hapten. A high yield of immunoglobulin G (IgG) against the azobenzene derivative took place in rabbits injected with azo-nanoparticles, whereas no increase in IgG was recognized in other rabbits treated solely with chemically equivalent azobenzene dye instead of azo-nanoparticles. Electron microscopy and surface plasmon resonance spectroscopy indicated that the IgG obtained specifically recognized the difference between the isomer conformations of the azobenzene moiety.     

Photomodulation of PS-modified oligonucleotides containing azobenzene substituent at pre-selected positions in phosphate backbone

A new protocol has been developed for incorporation of a photoisomerizable azobenzene moiety into synthetic stereo-enriched [R_p] and [S_p] PS-oligonucleotides. The azobenzene pendant is attached at pre-selected positions in internucleotidic phosphorothioate oligonucleotides of both [R_p] and [S_p] diastereomers using a novel reagent, N-iodoacetyl-p-aminoazobenzene, 1. The modified oligomers are purified on HPLC, characterized by LC–MS, and examined for their thermal and photoisomerization properties. The azobenzene moiety imparts greater stability to oligomer duplexes in (E) NN configuration as compared to (Z) configuration. The placement of the azobenzene pendant close to 5′-terminus (n − 1) and 3′-terminus of the modified PS-oligos contributes maximum stability to the duplex while a gradual decline in stability occurs with azobenzene moving toward middle of the duplex. Circular Dichroism studies reveal that the chiral environment at the phosphorus center of the PS-oligos does not alter the global conformation of the DNA duplex as such, suggesting conservation of conformation of the modified DNA strands.     

Copper-dependent DNA damage induced by hydrazobenzene, an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5'-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Synthesis of oligonucleotides containing a new azobenzene fragment with efficient photoisomerizability

The azobenzene derivatives possessing substituents of ROCH₂CH₂O- and-CH₂CH₂OR′ or -CONHCH₂CH₂OR′ at p,p′-positions, where R and R′ are 4,4′-dimethoxytrityl and 2-cyanoethyl-N,N′-diisopropylphophoramidite, have been synthesized for linking two oligonucelotide segments. It has been found that the azobenzene linkers efficiently undergo trans–cis isomerization by exposing to UV light. The conversion efficiency showed slight dependence on structure or conformation of oligonucleotides attached to the azobenzene chromophore. The cis-form of the azobenzene in oligonucleotides was sufficiently stable at low temperature under dark. The present findings would open the way for light switch of nucleic acid structures.     

Azobenzene Photoisomerization-Induced Destabilization of B-DNA

Molecular photoswitches provide a promising way for selective regulation of nanoscaled biological systems. It has been shown that conformational changes of azobenzene, one of the widely used photoswitches, can be used to reversibly control DNA duplex formation. Here, we investigate the conformational response of DNA upon azobenzene binding and isomerization, using a threoninol linker that has been experimentally investigated recently. To this end, nonequilibrium molecular dynamics simulations are carried out using a switching potential describing the photoinduced isomerization. Attachment of azobenzene leads to a distortion of the DNA helical conformation that is similar for the trans and cis forms. However, the trans form is stabilized by favorable stacking interactions whereas the cis form is found to remain flipped out of the basepair-stacked position. Multiple azobenzene attachment augments the distortion in DNA helical conformation. The distorted DNA retains nativelike pairing of bases at ambient temperatures, but shows weaker basepairing compared to native DNA at an elevated temperature.     

Synthesis and application of an azobenzene amino acid as a light-switchable turn element in polypeptides

The synthesis of an azobenzene amino acid (aa) for use as a photo-inducible conformational switch in polypeptides is described. The compound can be easily incorporated into an aa sequence by solid-phase peptide synthesis using standard 9-fluorenylmethoxycarbonyl methods. A reversible conformational change of the peptide backbone is induced by switching between the cis and trans configurations of the azobenzene moiety by irradiation with light of suitable wavelength. Thermal cis --> trans isomerization of this azobenzene aa is slow, enabling detailed structural investigations of the modified peptides, e.g., using NMR techniques. The total time for the synthesis of the photoswitch is typically 4 d, with an overall yield of 40-50%.     

Conformational changes of DNA by photoirradiation of DNA-bis(Zn(II)-cyclen)-azobenzene complex

Bis(Zn(II)-cyclen)-azobenzene derivative, which has two Zn(II)-macrocyclic tetraamine complexes connected through azobenzene spacer, has been synthesized as a cross-linking agent fordoublestranded DNA in aqueous solution. The Zn(II)-cyclen derivative selectively binds to A-T base pairs producing complexes between the Zn(II)-cyclen moiety and the imide-deprotonated thymine with breaking A-T base pairs. The azobenzene spacer undergoes cis/trans photoisomerization in the complex between the Zn(II)-cyclen derivative and the DNA duplex. The conformation of the DNA remarkably changed by photoisomerization of the azobenzene linker, when the Zn(II)-cyclen derivative binds to the DNA duplex with an interstrand cross-linking manner     

Photo-regulated duplex- and triplex-formation of the modified DNA carrying azobenzene.

The duplex- and triplex- forming activity of oligonucleotide was photo-regulated by using the isomerization of azobenzene in the side chain. When the azobenzene was isomerized from the trans-form to the cis-form by photo-irradiation, the melting temperatures of the duplex and triplex between the oligonucleotide and its complementary counterpart were significantly lowered.     

Photomodulation of conformational states. IV. Integrin-binding RGD-peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent

In previous studies we have investigated octapeptides backbone-cyclized by (4-amino)phenyl azobenzoic acid (APB) or (4-aminomethyl)phenylazobenzoic acid (AMPB) and containing the active-site sequence Cys-Ala-Thr-Cys-Asp from the thioredoxin reductase. The conformational and redox properties of these peptides were strongly dependent on the isomeric state of the azobenzene chromophore. Using the same approach we were successful in constructing photoresponsive ligands for alphavbeta3 integrin containing the Arg-Gly-Asp (RGD) sequence as binding motif. For achieving maximal conformational restriction of the peptide a reduced ring size compared to our previous azobenzene peptides was employed in the cyclic peptide c[Asp-D-Phe-Val-AMPB-Lys-Ala-Arg-Gly-]. Conformational properties of the trans and cis isomers of this peptide in solution were investigated by CD and NMR and were found to differ markedly from the thioredoxin derived azobenzene peptides. In a second peptide, c[Asp-D-Phe-Val-Lys-AMPB-Ala-Arg-Gly-], shifting the position of the chromophore lead to a marked decrease in affinity. With the availability of the x-ray structure of a cyclic RGD-pentapeptide bound to alphavbeta3 integrin (PDB entry 1L5G) modeling of possible bound conformations for trans and cis isomers of both azobenzene peptides was possible. Notably, both peptides in either isomeric form share the same overall conformation in the bound state according to our molecular dynamics simulations.     

Using azobenzene incorporated DNA aptamers to probe molecular binding interactions

The rational design of DNA/RNA aptamers for use as molecular probes depends on a clear understanding of their structural elements in relation to target-aptamer binding interactions. We present a simple method to create aptamer probes that can occupy two different structural states. Then, based on the difference in binding affinity between these states, target-aptamer binding interactions can be elucidated. The basis of our two-state system comes from the incorporation of azobenzene within the DNA strand. Azobenzene can be used to photoregulate the melting of DNA-duplex structures. When incorporated into aptamers, the light-regulated conformational change of azobenzene can be used to analyze how aptamer secondary structure is involved in target binding. Azobenzene-modified aptamers showed no change in target selectivity, but showed differences in binding affinity as a function of the number, position, and conformation of azobenzene modifications. Aptamer probes that can change binding affinity on demand may have future uses in targeted drug delivery and photodynamic therapy.     

Conformational Changes of DNA by Photoirradiation of DNA-Bis(ZnII-Cyclen)-Azobenzene Complex

Bis(Zn^II-cyclen)-azobenzene derivative, which has two Zn^II-macrocyclic tetraamine complexes connected through azobenzene spacer, has been synthesized as a cross-linking agent for double stranded DNA in aqueous solution. The Zn^II-cyclen derivative selectively binds to A-T base pairs producing complexes between the Zn^II-cyclen moiety and the imide-deprotonated thymine with breaking A-T base pairs. The azobenzene spacer undergoes cis/trans photoisomerization in the complex between the Zn^II-cyclen derivative and the DNA duplex. The conformation of the DNA remarkably changed by photoisomerization of the azobenzene linker, when the Zn^II-cyclen derivative binds to the DNA duplex with an interstrand cross-linking manner.     

Photo-regulation of DNA/RNA duplex formation by azobenzene-tethered DNA towards antisense strategy.

Modified DNA carrying an azobenzene was successfully applied to the photo-regulation of DNA/RNA hybridization. When the azobenzene was isomerized from trans- to cis-form on UV-irradiation, the melting temperature of the duplex was significantly lowered. This process was totally reversible so that the Tm increased by cis-->trans isomerization induced by visible light irradiation.     

Dynamic of the volume and structure of the soil bacterial complex in the presence of azobenzene

Azobenzene exerted no significant effect on the dynamics and the species composition of the saprophytic soil bacterial complex, which remained almost the same as in the control and was characterized by the predominance of Curtobacterium sp., Arthrobacter globiformis, and Bacillus megaterium in all stages of succession. Some heterotrophic bacteria were found to be able to accumulate azobenzene. Bac. cereus and Bac. polymyxa degraded azobenzene during their cultivation in nutrient media.     

Synthesis and Binding Properties of Oligonucleotides Containing an Azobenzene Linker

Abstract

Incorporation of an azobenzene-4,4′-diamide group via a linker arm into the 3′-hydroxyl function of one oligonucleotide segment and the 5′-OH of other oligonucleotide has been described. The binding of the oligonucleotides containing the azobenzene linker was investigated by UV melting behaviors. The azobenzene linker has been shown to be useful as an effective bridge for stabilizing hairpin duplex and triplex.     

Ochrobactrum intermedium ANKI, a nitrogen-fixing bacterium able to decolorize azobenzene

Morphological and biochemical properties of the nitrogen-fixing strain Ochrobactrum intermedium ANKI, intensely growing on media with azo compounds, and its resistance to various common xenobiotics were investigated. The kinetics of azobenzene conversion by O. intermedium ANKI was studied. Under cometabolism conditions, up to 40 mg of azobenzene per liter of medium were decolorized within one week. It was shown that the strain possessed molybdenum-dependent nitrogenase activity, and its nitrogenase system was sensitive to oxygen and fixed nitrogen in the medium.     

Micronucleus induction by azobenzene and 1,2-dibromo-3-chloropropane in the rat: evaluation of a triple-dose protocol

A triple-dose protocol was evaluated in the rat bone-marrow micronucleus test using azobenzene and 1,2-dibromo-3-chloropropane (DBCP) as test compounds. Dosing with 250 mg/kg azobenzene over 3 consecutive days led to an accumulation of micronucleated polychromatic erythrocytes. The magnitude of the effect was the same as that obtained after a single dose of 750 mg/kg in a previous study. In the single-dose study the effect was only detected at the 48 h sampling time. With the triple-dose protocol the effect was apparent 24 h after the last dose--thus eliminating the need for more than one sampling time. In the case of DBCP, the positive effect observed after a single dose diminished with increasing number of doses for the low dose but plateaued for the high dose, accompanied by a marked cytotoxic effect. It is therefore concluded, that for one of the compounds, azobenzene, the triple-dose protocol provided an advantage whereas for the other, DBCP, it failed to do so and led to results which are rather difficult to interpret. Furthermore, the number of animals necessary to select an appropriate dose level appears to be higher than in the single-dose protocol.     

Preparation and UV-protective properties of functional cellulose fabrics based on reactive azobenzene Schiff base derivative

Azobenzene Schiff base possesses excellent photochromic or thermochromic properties based on intermolecular proton transfer or cis-trans isomerization. The azobenzene Schiff base containing two reactive groups, N, N-bis{p-[(2′-sulphatoethyl)sulphonyl phenylazo] salicylidene}-1,2-ethylenediamine (BSPEA), was applied to modify cellulose materials. The functional cellulose fabrics containing azobenzene Schiff base groups were prepared. The chemical and morphological structures of functional cellulose fabrics were characterized by element analysis, FT-IR spectrum, and scanning electron microscopy (SEM). The UV-protection properties of the fabrics were investigated by the ultraviolet transmittance spectra and ultraviolet protection factor (UPF). The results show that the functional cellulose fabrics had excellent UV-protection properties with higher UPF value (UPF value reached 31.7) and lower ultraviolet transmittance (less than 5%). The modified cellulose fabrics had not significant influence on the physical properties. The functional cellulose fabrics based on reactive azobenzene Schiff base would have potential application in textile and functional materials.     

Synthesis of Photo-Responsive Acridine-Modified DNA and Its Application to Site-Selective RNA Scission

Photo-responsive phosphoramidite monomers, which bear an azobenzene between acridine and the phosphoramidite unit, were synthesized, and incorporated into oligonucleotides. Upon UV irradiation, the azobenzene in the modified DNA efficiently isomerized from the trans isomer into the cis isomer. Although the T_m values of their duplexes with complementary DNA were not much changed by the isomerization, site-selective RNA scission was significantly accelerated by the UV irradiation when Mn(II) ion was used as the catalyst for RNA scission.