Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin-biotin recognition.

We describe a method for the preparation of novel long (hundreds of nanometers), uniform, inter-molecular G4-DNA molecules composed of four parallel G-strands. The only long continuous G4-DNA reported so far are intra-molecular structures made of a single G-strand. To enable a tetra-molecular assembly of the G-strands we developed a novel approach based on avidin-biotin biological recognition. The steps of the G4-DNA production include: (i) Enzymatic synthesis of long poly(dG)-poly(dC) molecules with biotinylated poly(dG)-strand; (ii) Formation of a complex between avidin-tetramer and four biotinylated poly(dG)-poly(dC) molecules; (iii) Separation of the poly(dC) strands from the poly(dG)-strands, which are connected to the avidin; (iv) Assembly of the four G-strands attached to the avidin into tetra-molecular G4-DNA. The average contour length of the formed structures, as measured by AFM, is equal to that of the initial poly(dG)-poly(dC) molecules, suggesting a tetra-molecular mechanism of the G-strands assembly. The height of tetra-molecular G4-nanostructures is larger than that of mono-molecular G4-DNA molecules having similar contour length. The CD spectra of the tetra- and mono-molecular G4-DNA are markedly different, suggesting different structural organization of these two types of molecules. The tetra-molecular G4-DNA nanostructures showed clear electrical polarizability. This suggests that they may be useful for molecular electronics.

Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part III. Hydrogels as carriers for immobilization of proteins.

Four series of macroporous hydrogels based on crosslinked copolymers of 2-hydroxyethyl methacrylate (HEMA)-sodium methacrylate (MANa), copolymer HEMA-[2-(methacryloyloxy)ethyl]trimethylammonium chloride (MOETACl), terpolymer HEMA-MANa-MOETACl and on a polyelectrolyte complex were used as carriers for immobilization of proteins, chicken egg white albumin and avidin. The adsorption capacity of the hydrogels for the two proteins, kinetics and pH dependence of albumin adsorption and desorption were studied. The morphology of the hydrogels with and without immobilized albumin was studied by low-vacuum scanning electron microscopy.

Sensitive bondforce measurements of ligand-receptor pairs with magnetic beads.

The bondforces between biotinylated surfaces and streptavidin or avidin coated beads are investigated by a magnetic field based manipulation system for magnetic microbeads. The magnetic field is generated by currents through a set of conducting lines, and its gradient exerts a force onto the magnetic beads. The force can be increased until the bond between the bead and the surface breaks. Consistent with other groups we found two conformations for both investigated bonds. The measured bondforces for the two conformations are for Streptavidin-Biotin: 55.9 and 244.7 fN and for Avidin-Biotin: 15.9 and 58.4 fN. These very low bondforces (10-100 times smaller than earlier measurements) match to the extremely low loading rate of about 1 fN/s. This new technique thus allows to investigate biomolecular bonds by extremely low forces.

DNA condensation by high-affinity interaction with avidin.

Avidin, the basic biotin-binding glycoprotein from chicken egg white, is known to interact with DNA, whereas streptavidin, its neutral non-glycosylated bacterial analog, does not. In the present study we investigated the DNA-binding properties of avidin. Its affinity for DNA in the presence and absence of biotin was compared with that of other positively charged molecules, namely the protein lysozyme, the cationic polymers polylysine and polyarginine and an avidin derivative with higher isoelectric point (pI approximately 11) in which most of the lysine residues were converted to homoarginines. Gel-shift assays, transmission electron microscopy and dynamic light scattering experiments demonstrated an unexpectedly strong interaction between avidin and DNA. The most pronounced gel-shift retardation occurred with the avidin-biotin complex, followed by avidin alone and then guanidylated avidin. Furthermore, ultrastructural and light-scattering studies showed that avidin assembles on the DNA molecule in an organized manner. The assembly leads to the formation of nanoparticles that are about 50-100 nm in size (DNA approximately 5 kb) and have a rod-like or toroidal shape. In these particles the DNA is highly condensed and one avidin is bound to each 18 +/- 4 DNA base pairs. The complexes are very stable even at high dilution ([DNA] =10 pM) and are not disrupted in the presence of buffers or salt (up to 200 mM NaCl). The other positively charged molecules also condense DNA and form particles with a globular shape. However, in this case, these particles disassemble by dilution or in the presence of low salt concentration. The results indicate that the interaction of avidin with DNA may also occur under physiological conditions, further enhanced by the presence of biotin. This DNA-binding property of avidin may thus shed light on a potentially new physiological role for the protein in its natural environment.

ATP synthase from Saccharomyces cerevisiae: location of the OSCP subunit in the peripheral stalk region.

A biotinylation signal has been fused to the C terminus of the oligomycin sensitivity conferral protein (OSCP) of the ATP synthase complex from Saccharomyces cerevisiae. The signal is biotinylated in vivo and the biotinylated complex binds avidin in vitro. By electron microscopy of negatively stained particles of the ATP synthase-avidin complex, the bound avidin has been localised close to the F(1) domain. The images were subjected to multi-reference alignment and classification. Because of the presence of a flexible linker between the OSCP and the biotinylation signal, the class-averages differ in the position of the avidin relative to the F(1) domain. These positions lie on an arc, and its centre indicates the position of the C terminus of the OSCP on the surface of the F(1) domain. Since the N-terminal region of the OSCP is known to interact with the N-terminal regions of alpha-subunits, which are on top of the F(1) domain distal from the F(o) membrane domain, the OSCP extends almost 10nm along the surface of F(1) down towards F(o) where it interacts with the C terminus of the b subunit, which extends up from F(o). The labelling technique has also allowed a reliable 2D projection map to be developed for the intact ATP synthase from S.cerevisiae. The map reveals a marked asymmetry in the F(o) part of the complex that can be attributed to subunits in the F(o) domain.

Detección del antígeno viral de la rabia por el método avidina - biotina - peroxidasa / Detection of the rabies viral antigen by the avidin - biotin - peroxidase method

Antecedentes: La rabia humana y animal es frecuente en Colombia. Su diagnóstico requiere inmnofluorescencia directa (IFD) e inoculación intracerebral al ratón (IIC), condiciones que alcanzan pocos laboratorios. La histopatología muestra cuerpos de Negri entre el 40 por ciento al 87 por ciento de los casos. Nuestro objetivo es desarrollar y determinar la utilidad del método avidina-biotina-peroxidasa (ABP) en tejido fijado en formol e incluido en parafina. Materiales y métodos: Se estandarizó la técnica ABP usando muestras de encéfalo con rabia confirmada por IFD, IIC al ratón o histopatología. La técnica se pudo a prueba estudiando cortes de 100 encéfalos humanos o de animales sospechosos de tener rabia. Se escogieron encéfalos incluídos en parafina o mantenidos en congelación durante años, SNC fresco de ratón inoculado con virus fijo y cerebros normales o con otras encefalitis virales. Se determinó la sensibilidad, la especificidad y la reproducibilidad del método. Resultados: La técnica de ABP muestra sensibilidad mayor o igual a .80 y especificidad mayor o igual a .97. La concordancia con respecto a la IIC y a la IFD dio resultados entre substanciales y casi perfectos, según la escala de Landys y Koch, niveles altos que también se obtienen al evaluar la reproducibilidad intraobservador e interobservador. La técnica fue siempre mejor que la HE. Conclusiones: La técnica es útil, confiable, reproducible, fácil de realizar y funciona bien inclusive en encéfalos con pobre preservación tisular. Merece incluirse entre los procedimientos de rutina o alternativos en el dignóstico de la rabia humana y animal

Two-dimensional crystallization of avidin on biotinylated lipid monolayers.

Two-dimensional crystals of avidin were obtained on mixed lipid monolayers containing biotinylated lipids (N-biotinyl-dipalmitoyl-L-alpha-phosphatidyl ethanolamine and dioleoyl phosphatidyl choline) by specific interaction. Image analysis of electron micrographs of these crystals revealed p2 symmetry with the unit cell parameters a = 66 +/- 2 A, b = 68 +/- 1 A, and gamma = 121 +/- 4 degrees. The projection map showed, at a resolution of about 27 A, that the four subunits within one avidin molecule are separated into two parts. Comparison between avidin and streptavidin reveals that avidin molecule binds to the lipid monolayer in an orientation similar to that of streptavidin.