Streptavidin-coated Surface Research Articles

τ Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains: DOMAIN IV, LOCATED WITHIN THE UNIQUE C TERMINUS OF τ, BINDS THE REPLICATION FORK HELICASE, DnaB

Interaction between the tau subunit of the DNA polymerase III holoenzyme and the DnaB helicase is critical for coupling the replicase and the primosomal apparatus at the replication fork (Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643-650). In the preceding manuscript, we reported the identification of five putative structural domains within the tau subunit (Gao, D., and McHenry, C. (2000) J. Biol. Chem. 275, 4433-4440). As part of our systematic effort to assign functions to each of these domains, we expressed a series of truncated, biotin-tagged tau fusion proteins and determined their ability to bind DnaB by surface plasmon resonance on streptavidin-coated surfaces. Only tau fusion proteins containing domain IV bound DnaB. The DnaB-binding region was further limited to a highly basic 66-amino acid residue stretch within domain IV. Unlike the binding of immobilized tau(4) to the DnaB hexamer, the binding of monomeric domain IV to DnaB(6) was dependent upon the density of immobilized domain IV, indicating that DnaB(6) is bound by more than one tau protomer. This observation implies that both the leading and lagging strand polymerases are tethered to the DnaB helicase via dimeric tau. These double tethers of the leading and lagging strand polymerases proceeding through the tau-tau link and an additional tau-DnaB link are likely important for the dynamic activities of the replication fork.

Inhibition of p53-dependent transcription by BOX-I phospho-peptide mimetics that bind to p300.

The N-terminal BOX-I domain of p53 containing a docking site for the negative regulator MDM2 and the positive effector p300, harbours two recently identified phosphorylation sites at Thr18 or Ser20O whose affect on p300 is undefined. Biochemical assays demonstrate that although MDM2 binding is inhibited by these phosphorylations, p300 binding is strikingly stabilized by Thr18 or Ser20 phosphorylation. Introducing EGFP-BOX-I domain peptides with an aspartate substitution at Thr18 or Ser20 induced a significant inhibition of endogenous p53-dependent transcription in cycling cells, in irradiated cells, as well as in cells transiently co-transfected with p300 and p53. In contrast an EGFP-wild-type BOX-I domain peptide stimulated p53 activity via inhibition of MDM2 protein binding. These results suggest that phosphorylation of p53 at Thr18 or Ser20 can activate p53 by stabilizing the p300-p53 complex and also identify a class of small molecular weight ligands capable of selective discrimination between MDM2- and p300-dependent activities.

Analysis of the RNase T1 mediated cleavage of an immobilized gapped heteroduplex via fluorescence correlation spectroscopy.

We report a new method for studying the activity of hydrolytic enzymes. Fluorescence correlation spectroscopy was used to observe online the hydrolyzation of a rhodamine B-labeled substrate by ribonuclease T1. A gapped heteroduplex substrate - a hybrid of a ribooligonucleotide and two smaller complementary deoxyribooligonucleotides - was immobilized via biotin to a streptavidin-coated surface of a coverslip. The reported method opens the possibility to study the cleavage of small substrates differing only slightly in molecular weight from the enzyme reaction product. The use of fluorescence correlation spectroscopy allows the detection of very low enzyme concentrations (down to 10(-21) mol 0.05 fM of RNase T1, corresponding to about 600 RNase T1 molecules in 0.02 ml).

On Death Numbers and Survival Times of Single Dye Molecules

In fluorescence measurements on the single molecule level the photochemical stability of the fluorophore is an important factor. We measured in 102 cases the photodestruction of individual tetramethylrhodamine molecules linked to a 217-bp DNA sequence which was attached to a streptavidin-coated glass surface. The fluorophores were excited by a green HeNe laser at 543.5 nm. For each molecule the survival time (time during which fluorescence can be observed), the fluorescence intensity, and the death number (number of detected photons before decomposition) were registered. On the basis of the analysis of single molecule observations, an exponential distribution of the death numbers and survival times of the 102 molecules was found.

DNA-Directed Immobilization: Efficient, Reversible, and Site-Selective Surface Binding of Proteins by Means of Covalent DNA–Streptavidin Conjugates

Covalent DNA–streptavidin conjugates have been utilized for the reversible and site-selective immobilization of various biotinylated enzymes and antibodies by DNA-directed immobilization (DDI). Biotinylated alkaline phosphatase, β-galactosidase, and horseradish peroxidase as well as biotinylated anti-mouse and anti-rabbit immunoglobulins have been coupled to the DNA–streptavidin adapters by simple, two-component incubation and the resulting preconjugates were allowed to hybridize to complementary, surface-bound capture oligonucleotides. Quantitative measurements on microplates indicate that DDI proceeds with a higher immobilization efficiency than conventional immobilization techniques, such as the binding of the biotinylated proteins to streptavidin-coated surfaces or direct physisorption. These findings can be attributed to the reversible formation of the rigid, double-stranded DNA spacer between the surface and the proteins. Moreover, BIAcore measurements demonstrate that DDI allows a reversible functionalization of sensor surfaces with reproducible amounts of proteins. Ultimately, the simultaneous immobilization of different compounds using microstructured oligonucleotide arrays as immobilization matrices demonstrate that DDI proceeds with site selectivity due to the unique specificity of Watson–Crick base pairing.

A Scintillating Microplate Assay for the Assessment of Protein Kinase Activity

Protein kinases, a class of enzymes that phosphorylate certain tyrosine, serine, and threonine residues, play an important role in cellular functions and are important targets in drug discovery research. Thus, it is of interest to develop a simple assay that can be used to measure protein kinase activity toward specific substrates and is suitable for the high throughput screening (HTS) of potential kinase inhibitors. The scintillation proximity concept has been successfully applied for measuring specific kinase activity using surfaces passively coated with a peptide substrate. In this study, we evaluated kinase assay performance on three ScintiStrip platforms: unmodified surface, streptavidin-coated surface, and streptavidin covalently attached to surface. The high affinity of streptavidin toward biotin-linked peptide substrates makes it a unique platform for measuring specific incorporation of radiolabeled phosphate into selected substrates of specific enzymes in the presence of others. Therefore, this assay may be used with cell extracts containing impure kinases as well as with purified enzymes. The scope of this assay was demonstrated with purified tyrosine kinases (e.g., p60c-src kinase) and A431 cell extracts. This scintillation proximity assay is universal, simple, rapid, accurate, and can be adapted for use with robotics for HTS.

New Immunoelectrochemiluminometric Assay to Measure Serum Thyrotropin,

The log/linear relation between serum thyrotropin (TSH) and free thyroxine (FT4) makes TSH, when measured by a highly sensitive method, a more sensitive indicator of thyroid disfunction than FT4 (1). Nonisotopic immunometric assays are the methods of choice to measure serum TSH, as they are at least equally sensitive to IRMAs, can be automated, are generally faster and more convenient than IRMAs, and do not use radioisotopes. Within the former group of methods, the immunochemiluminometric assay is increasingly popular because of its inherent sensitivity, but instrumental and triggering modes may jeopardize it. A new immunoelectrochemiluminometric assay (IECMA) has recently come on the market (2). It is based on electrogenerated chemiluminescence that, aiming to avoid the instability of luminophores, does not proceed in free solution but on to the surface of a platinum electrode (3)(4). We describe the evaluation of such a method using the random access analyzer Elecsys® 2010 (Boehringer Mannheim Diagnostics). The method consists of two immunological “sandwich” reactions linking, at one side, sample TSH to a solid phase of paramagnetic streptavidin-coated microparticles by means of a biotinylated monoclonal TSH-specific antibody and, at the other side, a Ru-labeled TSH-specific antibody. Separation of bound and unbound TSH is made by a magnet, and luminescence from a luminophore is triggered electrochemically. The immunological reaction consists of a first incubation where 50 μL of sample, the biotinylated antibody, and the Ru-labeled antibody react for 9 min, when antibodies capture the TSH present in the sample to form a sandwich complex. In a second step, streptavidin-coated paramagnetic microparticles are added, and during a second 9-min incubation time the biotinylated antibody attaches to the streptavidin-coated surface of the microparticles. The reaction mixture …

Conformational fluctuations in single DNA molecules.

Measurement of fluorescent lifetimes of dye-tagged DNA molecules reveal the existence of different conformations. Conformational fluctuations observed by fluorescence correlation spectroscopy give rise to a relaxation behavior that is described by "stretched" exponentials and indicates the presence of a distribution of transition rates between two conformations. Whether this is an inhomogeneous distribution, where each molecule contributes with its own reaction rate to the overall distribution, or a homogeneous distribution, where the reaction rate of each molecule is time-dependent, is not yet known. We used a tetramethylrhodamine-linked 217-bp DNA oligonucleotide as a probe for conformational fluctuations. Fluorescence fluctuations from single DNA molecules attached to a streptavidin-coated surface directly show the transitions between two conformational states. The conformational fluctuations typical for single molecules are similar to those seen in single ion channels in cell membranes.

Direct observation of aminoglycoside-RNA interactions by surface plasmon resonance.

The specificity of neomycin B and related aminoglycoside antibiotics in their interaction with the Rev responsive element (RRE) of HIV-1 mRNA has been studied by directly observing the aminoglycoside-RNA complexes using surface plasmon resonance. Several different RNA sequences, each with a biotin tag, have been prepared using T7 RNA polymerase-catalyzed transcription of synthetic DNA templates and have been immobilized on a streptavidin-coated surface for the binding study. The results indicate that neomycin B is not specific for the G-rich bubble region in RRE. Rather, it appears to interact with three different sites, each with a submicromolar dissociation constant, within the 67-nucleotide domain II of RRE. Further analysis of neomycin B binding with three short synthetic RNA hairpins showed binding with submicromolar affinity and 1:1 stoichiometry in each case. This suggests that neomycin B may generally bind with this affinity to regular A-form RNA or hairpin loops. The approach described here is generally useful for understanding the fundamental interactions involved in the specific recognition of nucleic acids by small molecules which is the basis of rational drug design.

Scanning probe microscopy of biomolecules and polymeric biomaterials

Critical problems associated with the analysis of biological macromolecules and polymeric biomaterials by scanning probe microscopy are identified. The problems of molecular immobilisation and distribution and data validation are discussed. A number of solutions are described, including the application of a conductive coating and the covalent attachment of biomolecules to suitable substrates. Recent work in the authors' laboratory is described. The conductive coating approach has been employed to study helical macromolecules (including polysaccharides and DNA) and protein molecules. A variety of covalent attachment procedures have been examined, including the adsorption of thiolated proteins onto gold surfaces, the coupling of proteins to self-assembled monolayers, and the attachment of biotinylated molecules to streptavidin-coated polystyrene surfaces. Studies of polymeric biomaterials are also described. Scanning tunnelling microscopy and transmission electron microscopy have been compared in studies of poly(ethylene oxide), and the interactions of ferritin with a ferritin antibody immobilised on a polystyrene surface have been examined.

Detection and kinetic studies of triplex formation by oligodeoxynucleotides using real-time biomolecular interaction analysis (BIA).

Real-time biomolecular interaction analysis (BIA) has been applied to triplex formation between oligodeoxynucleotides. 5'-Biotinylated oligonucleotides were immobilised on the streptavidin-coated surface of a biosensor chip and subsequently hybridised to their complementary strand. Sequence-specific triplex formation was observed when a suitable third-strand oligopyrimidine was injected over the surface-bound duplex. In addition, a single-stranded oligonucleotide immobilised on the chip surface was able to capture a DNA duplex by triplex recognition. The presence of spermine increases the rate of association between the third strand and immobilised duplex, but at elevated spermine concentrations non-specific association is observed. A preliminary kinetic analysis of triplex formation at pH 5.2 by an 11mer third strand containing thymine, cytosine and uracil is reported. Values for the association and dissociation rate constants were determined to be (1.9 +/- 0.2) x 10(3) M-1 s-1 and (8.1 +/- 1.9) x 10(-5) s-1, respectively.

Monitoring of oligonucleotide hybridization using light-addressable potentiometric and evanescent wave fluorescence sensing

Hybridized pairs of oligonucleotides, one biotinylated and the other FITC-labeled, were captured on streptavidincoated surfaces via biotin-streptavidin binding. Detection of the fluoresceinated probe was achieved (1) potentiometrically by light-addressable potentiometric sensing (LAPS) using urease-conjugated anti-fluorescein antibodies and (2) by evanescent wave spectroscopy (EWS) using light internally reflected along a quartz fiber on which the hybrid duplexes were captured facilitating excitation of the FITC-labeled probe in the evanescent wave zone. As little as 20 pg fluoresceinated oligonucleotide could be detected using the LAPS, whereas, the limit of detection using EWS was 50 ng. A dose-dependent reduction of signal was observed in the presence of a nonfluoresceinated oligonucleotide of identical sequence as that of the fluoresceinated target using both methods. Specificity of hybridization was confirmed by the ineffectiveness of added genomic DNA from several species in suppressing the signal. These data indicate that the LAPS and EWS methodologies may be useful for rapid and sensitive detection of oligonucleotides of defined sequence.

Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface.

Kinesin, an ATP-dependent microtubule motor, can be studied in vitro in motility assays where the kinesin is nonspecifically adsorbed to a surface. However, adsorption can inactivate kinesin and may alter its reaction kinetics. We therefore prepared a biotinated kinesin derivative, K612-BIO, and characterized its activity in solution and when bound to streptavidin-coated surfaces. K612-BIO consists of the N-terminal 612 amino acids of the Drosophila kinesin alpha subunit linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotination of the protein. We expressed K612-BIO at high levels using the baculovirus expression vector system and purified it to near-homogeneity. The expressed protein is completely soluble, and > 90% is bound by streptavidin. K612-BIO steady-state ATPase kinetics (KM,ATP = 24 microM, K0.5, microtubule = 0.61 mg ml-1, Vmax = approximately 25 s-1 head-1, 25 degrees C) are similar to those reported for intact kinesin. ATPase kinetics are not affected by the addition of streptavidin. Enzyme bound to a surface coated with streptavidin drove microtubule gliding in the presence of 2 mM ATP at 750 +/- 130 nm s-1 (26 degrees C). Activity was abolished by pretreatment of the surface with biotin, indicating that the microtubule movements are due to specifically bound enzyme. Motility assays based on specific attachment of biotinated enzyme to streptavidin-coated surfaces will be useful for quantitative analysis of kinesin motility and may provide a way to detect activity in kinesin derivatives or kinesin-like proteins that have not yet been shown to move microtubules.