Labelling of live cells using fluorescent aptamers: binding reversal with DNA nucleases

A reversible cell labelling method has been developed for non-destructive and non-invasive cell labelling and purification. Our method uses high affinity single strand DNA (ssDNA) aptamers against surface exposed target molecules on cells. The aptamers are subsequently removed from the cell surface using DNase nuclease treatment. We exemplified our method by labelling human acute lymphoblastic leukemia cells with Qdot-ssDNA aptamers, and restoring them to the label-free condition by treatment with Benzonase. Binding of the fluorescent-aptamers to the cells was evaluated by measuring fluorescence intensity and was further confirmed using flow cytometry. Removal of the aptamers can be achieved in ~10 min by the DNase nuclease digestion. Incubation of cells with aptamers or with the nucleases results in no apparent damage to the cells and does not affect their growth rates. The latter were equivalent to the rates measured for the untreated cells. Our method provides an alternative to traditional antibody-based techniques and could be especially suitable for non-invasive reversible cell labelling and cell separations where maintaining native cell activity is needed.     

Analysis of the structure and function of creatine kinase active sites using affinity modification

Data of studies of creatine kinase from rabbit skeletal muscle (EC 2.7.3.2) by affinity labelling and affinity chromatography are reviewed. Efficiencies of these techniques are demonstrated for analysis of cooperative interactions of the enzyme's active sites, nature of non-equivalence of enzyme subunits, distances between active sites which are situated on different subunits, dynamics of enzyme-substrate interactions and usefulness of affinity labelling for localization of amino acid residues in the enzyme active sites.     

Use of the biotin-avidin system for labelling, isolation and characterization of neural cell-surface proteins.

We describe a method for the selective labelling, isolation and electrophoretic analysis of cell-surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish-peroxidase-coupled or 35S-labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin-agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated.     

Concepts and approaches towards understanding the cellular redox proteome

The physiological activity of a significant subset of cell proteins is modified by the redox state of regulatory thiols. The cellular redox homeostasis depends on the balance between oxidation of thiols through oxygen and reactive oxygen species and reduction by thiol-disulfide transfer reactions. Novel and improved methodology has been designed during recent years to address the level of thiol/disulfide regulation on a genome-wide scale. The approaches are either based on gel electrophoresis or on chromatographic techniques coupled to high end mass spectrometry. The review addresses diagonal 2D-SDS-PAGE, targeted identification of specific redox-interactions, affinity chromatography with thioredoxins and glutaredoxins, gel-based and non-gel based labelling techniques with fluorophores (such as Cy3, Cy5, ICy), radioisotopes, or with isotope-coded affinity tags (ICAT), differential gel electrophoresis (DIGE) and combined fractional diagonal chromatography (COFRADIC). The extended methodological repertoire promises fast and new insight into the intricate regulation network of the redox proteome of animals, bacteria, and plants.     

Epoxide hydrolase is a marker for the smooth endoplasmic reticulum in rat liver.

Epoxide hydrolase (EH, EC 3.3.2.3) was chosen as a potential marker for smooth endoplasmic reticulum, because this enzyme is inducible by drugs such as phenobarbital. The hypothesis was verified in rat liver using immunochemical and immunocytochemical techniques. Antibodies were raised to the purified protein. These antibodies were affinity purified using the enzyme immobilized on Sepharose Ultrogel. The specificity of the antibodies was assayed by immunoelectrotransfer (Western blot). The labelling of rat liver thin frozen sections with protein A-gold particles demonstrated that the antibodies specifically recognised smooth endoplasmic reticulum membranes. Rough endoplasmic reticulum, other intracellular organelles and plasma membrane were unlabelled.     

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of ³²P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed.These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques.The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.     

Affinity labelling of human transcortin

The binding site of transcortin has been studied by using bromoacetyltestosterone and bromoacetylated derivatives of progesterone which were monohydroxylated at different positions of the steroid nucleus. Specificity of affinity labelling was demonstrated by the displad cortisol analog was added to a [3H]cortisol-transcortin complex solution. The binding site crevice was found to be very narrow in the vicinity of the A and B rings of steroid since 2alpha-hydroxyprogesterone, 6alpha- or 6beta-bromoacetoxyprogesterone and dexamethasone could not displace bound cortisol. A specific affinity labelling was obtained with 11alpha-bromoacetoxyprogesterone, 16alpha-bromoacetoxyprogesterone and 17beta-bromoacetyltestosterone. The results of the affinity labelling by these hormone analogs suggested that one methionine and one histidine residues were located within the active site:methionine might interact with the 11beta-hydroxyl group and histidine with the 20 keto group of cortisol.     

An investigation into the ATP requirement for phosphorylation of thylakoid proteins and for the ATP-induced decrease in the yield of chlorophyll fluorescence in chloroplasts at different stages of development

The phosphorylation of thylakoid membrane polypeptides has been investigated in chloroplasts prepared from peas that had been grown under intermittent light and then exposed to between 4 and 48 h of continuous light. At 4 h, when the ratio of the total amount of labelling of a 9 kDa-polypeptide relative to light-harvesting chlorophyll protein (LHCP) polypeptides was much greater than 1, the affinity for ATP was found to be the same (S_0.5, approx. 100 μM) for both polypeptides. In contrast, in fully greened chloroplasts, when labelling of LHCP was much greater than that of the 9 kDa-polypeptide, the S_0.5 for ATP was 40 μM for LHCP and 500 μM for the 9 kDa-polypeptide. A correlation was observed during development between the affinity for ATP of the 9 kDa-species and its abundance relative to LHCP. It is suggested that these polypeptides compete for phosphorylation by the same protein kinase. Simultaneous assay of the ATP-induced fluorescence decrease at different ATP concentrations revealed a close correlation with LHCP labelling but not with labelling of the 9 kDa-polypeptide. This correlation held irrespective of which polypeptide was the major phosphoprotein.     

RNA-binding proteins of Rana temporaria frog oocytes. Isolation by methods of affinity chromatography and radioactive labelling in vitro]

A fraction of RNA-binding proteins from the maturating frog oocytes of Rana temporaria was isolated by the method of affinity chromatography on poly-U coupled to Sepharose. These proteins were labelled with high activity by the method of reductive methylation using 3H-sodium borohydride. It was shown that during the isolation and labelling there is no loss or inactivation of any RNA-binding protein fraction which is essential for informosome-like particles formation. The molecular masses of polypeptide chains in the isolated RNA-binding protein fraction were determined.     

FISH and immunocytochemistry: towards visualising single target molecules in living cells

Knowledge of how molecules interact in space and time is crucial for understanding cellular processes. A host of novel techniques have been developed for the visualisation of single target molecules in living cells, many based on fluorescence in situ hybridisation (FISH) or immunocytochemistry (IC). To extend the applicability of FISH to living cells, special backbone-modified probes and specific conformations (molecular beacons) have been designed. In the case of IC, conventional immunoreagents have been fine-tuned with respect to size and affinity or replaced with new protein scaffolds based on ankyrin repeat proteins. Other key advances include the use of proximity ligation to confirm vicinity binding and the use of quantum dots, which have proven potential for cellular labelling.     

Catalytic properties of acetylcholinesterase, modified by N,N-dimethyl-2-phenylaziridinium. An alkane sulfonation reaction

By means of affinity labelling with N,N-dimethyl-2-phenylaziridinium ion (DPA) two forms of acetylcholinesterase were synthesized that contained one or two molecules of the label covalently attached to the enzyme. The reaction of native and covalently modified acetylcholinesterases with n-alkane sulfonyl chlorides CnH2n + 1SO2Cl at n = 1 -4 was used to characterize the reactivity and properties of the enzymes. It was found that labelling of acetylcholinesterase with one molecule of DPA did not affect the enzyme's reactivity. Acetylcholinesterase containing two labels (the second one presumably located at the anionic centre of the enzyme) displayed enhanced and more specific reactivity towards alkane sulfonyl chlorides. It was found that the phenomenon of acceleration caused by affinity modification is analogous to the influence of n-tetraalkylammonium ions on the same reaction. Therefore, the mechanism of regulation of the properties of the esteric centre, caused by affinity labelling of the enzyme at the anionic centre, is the same as in the case of n-tetralkylammonium ions.     

Comparison of ATP-pyrophosphate exchange reactions, catalyzed by two different "one-site" forms of tryptophanyl-tRNA-synthetase and by native enzyme

Kinetic parameters of the reaction of ATP-PPi exchange, catalysed by "one-site" form of beef pancreas tryptophanyl-tRNA-synthetase are determined. The one-site form was obtained by affinity labelling of one active centre of the enzyme with alkylated analog of tryptophan. The comparison of kinetic parameters of this one-site form with the other one produced earlier by affinity labelling with analog of tryptophanyl-tRNA and the native enzyme was carried out. It was shown that the presence of tRNA improves the discrimination properties of the enzyme and diminishes the concentrations of "dead-end" complexes. Negative cooperativity in binding of two molecules of tryptophan is observed, which disappears in the presence of ATP, leading to an apparent synergistic effect. The molecule of tryptophan, which is present in the active centre, diminishes affinity of pyrophosphate.     

Detection, using fluorescent lectins, of specific carbohydrate chains in primordial germ cells of anuran amphibians

A study of primordial germ cells (PGC) of Amphibia Anura was carried out after treatment of sections by different fluorescein isothiocyanate conjugated lectins (FITC-lectins). Specific labelling on the PGC is obtained with lectins, the activity of which is inhibited by D-galactose or N-acetyl-galactosamine. These osidic groups appear to be located more specifically on the PGC. The same labelling pattern is not obtained with lectins possessing major affinity for mannose, glucose, fucose and N-acetyl-glucosamine. Furthermore, changes in labelling pattern are observed during migration of PGC. It is suggested that D-galactose and N-acetyl-galactosamine might be related to membrane activity of PGC during migration. Ultrastructural study of the visualization of cell surface carbohydrates supplies some information on the localisation of these lectins receptors.     

Studies of the functional topography of Escherichia coli RNA polymerase. Affinity labelling of RNA polymerase in a promoter complex by phosphorylating derivatives of primer oligonucleotides

Amidation of the 5'-phosphate group of the heptanucleotide pdApdApdApdTpdCpdGprC and of its derivatives of the general formula (pdN)npdGprC (n = 0-5) with imidazole, N-methylimidazole, and 4-dimethylaminopyridine afforded a series of phosphorylating affinity reagents. The parent oligonucleotides of this series complementary to promoter A2 of T7 phage over the region (-5 to +2) are known to be efficient primers of the synthesis of RNA by Escherichia coli RNA polymerase with promoter A2 as template. Treatment of the complex RNA-polymerase X promoter-A2 with affinity reagents followed by addition of [alpha-32P]UTP resulted in labelling of RNA polymerase by the residues -(pdN)npdGprCprU (p = radioactive phosphate). This affinity labelling was highly selective because elongation of the covalently bound residues (pdN)npdGprC by prU residues was catalyzed by the active center of RNA polymerase. The most efficient reagents were N-methylimidazolides. A dramatic change of the pattern of labelling of the subunits beta, beta', and sigma took place with changing n. Maximum labelling of the beta subunit occurred at n = 1 and of the sigma subunit at n = 5. The targets in both the subunits were His residues. The alpha subunit was not specifically labelled.     

Self-assembly of proteins and their nucleic acids

We have developed an artificial protein scaffold, herewith called a protein vector, which allows linking of an in-vitro synthesised protein to the nucleic acid which encodes it through the process of self-assembly. This protein vector enables the direct physical linkage between a functional protein and its genetic code. The principle is demonstrated using a streptavidin-based protein vector (SAPV) as both a nucleic acid binding pocket and a protein display system. We have shown that functional proteins or protein domains can be produced in vitro and physically linked to their DNA in a single enzymatic reaction. Such self-assembled protein-DNA complexes can be used for protein cloning, the cloning of protein affinity reagents or for the production of proteins which self-assemble on a variety of solid supports. Self-assembly can be utilised for making libraries of protein-DNA complexes or for labelling the protein part of such a complex to a high specific activity by labelling the nucleic acid associated with the protein. In summary, self-assembly offers an opportunity to quickly generate cheap protein affinity reagents, which can also be efficiently labelled, for use in traditional affinity assays or for protein arrays instead of conventional antibodies.     

Highly selective labeling of the E. coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity

A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling. The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.     

Ultrastructural identification of apoptotic nuclei using the TUNEL technique

Summary We describe an ultrastructural adaptation of the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) for the identification of DNA fragmentation. Thin sections of tissue embedded in hydrophilic resin were nick end labelled with biotinylated dUTP which was subsequently labelled with avidin conjugated to gold particles. The technique was validated by labelling the nuclei of L929-8 cells treated with tumour necrosis factor α. These cells are known to respond to treatment with the factor by undergoing apoptosis. The method was then used on tissue from the chick embryo which is known to be undergoing programmed cell death. This tissue was from the neural tube and the posterior necrotic zone of the limb bud, where cells can be identified as undergoing apoptosis based on the morphology of their nuclei. The method specifically labelled heterochromatin adjacent to the nuclear envelope as well as the associated with the nucleolus of cells from regions of the embryo where programmed cell death was expected. In addition to labelling the nuclei of cells that were clearly undergoing apoptosis, the method also identified nuclei of apparently normal cells. This method, used in conjunction with corroborating techniques, provides a means for the early detection of cells undergoing DNA fragmentation, before the onset of gross apoptotic morphology, and in cells that do not show classical apoptotic characteristics.