Bridging the electrochemical and biological worlds with hybrid nanocomposites

Recent discoveries arising from a combination of the biological, physical, chemical and materials sciences have resulted in the invention of numerous hybrid molecules that possess strengths inherent to each individual discipline. Nanocomposites that link biological molecules to inorganic moieties have led to a family of new reagents with unique capabilities for cellular imaging and macromolecule detection. A recent report has extended the applications of these hybrid molecules from their use as detection and scaffolding reagents into the realm of a biologically functional molecule.     

Influence of linker unit on performance of palladium(II) coproporphyrin labelling reagent and its bioconjugates

In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin‐I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies. Copyright © 2003 John Wiley & Sons, Ltd.     

Cleavable linkers in chemical biology

Interest in cleavable linkers is growing due to the rapid development and expansion of chemical biology. The chemical constrains imposed by the biological conditions cause significant challenges for organic chemists. In this review we will present an overview of the cleavable linkers used in chemical biology classified according to their cleavage conditions by enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, oxidizing reagents.     

Picoinjection of Microfluidic Drops Without Metal Electrodes

Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of these electrodes adds cumbersome and error-prone steps to the device fabrication process. We have developed a technique that obviates the needs for metal electrodes during picoinjection. Instead, it uses the injection fluid itself as an electrode, since most biological reagents contain dissolved electrolytes and are conductive. By eliminating the electrodes, we reduce device fabrication time and complexity, and make the devices more robust. In addition, with our approach, the injection volume depends on the voltage applied to the picoinjection solution; this allows us to rapidly adjust the volume injected by modulating the applied voltage. We demonstrate that our technique is compatible with reagents incorporating common biological compounds, including buffers, enzymes, and nucleic acids.     

Proteomic analyser with applications to diagnostics and vaccines

This paper describes a method for proteomic analysis with applications to diagnostics and vaccines. A panel of N (> or = 1) reagents called X(j), with j = 1 to N, is used. The binding strength of each of the X(j) reagents to each other is measured, for example by an ELISA assay, giving an N x N matrix K. The matrix K is used to define another set of N reagents called Y(j), with j = 1 to N, each of which is a linear combination of the X(j) reagents and each of which is tailored to be complementary to one of the X(j) reagents. Each of the N pairs of reagents X(j) and Y(j) defines an axis in an N-dimensional shape space. The definition of these axes facilitates proteomic analysis of diverse biological samples, for example, mixtures of proteins such as serum samples or T cell extracts. A method for defining and measuring similarity between pairs of biological samples and between sets of biological samples in the context of the set of N reagent pairs is described. This leads to methods for using the N reagent pairs in the diagnosis of diseases and in the formulation of preventive and therapeutic vaccines. The relationship of this work to previous research on shape space is discussed.     

Recombinant antibodies: a new reagent for biological agent detection

Antibodies are critical reagents used in several biodetection platforms for the identification of biological agents. Recent advances in phage display technology allow isolation of high affinity recombinant antibody fragments (Fabs) that may bind unique epitopes of biological threat agents. The versatility of the selection process lends itself to efficient screening methodologies and can increase the number of antigen binding clones that can be isolated. Pilot scale biomanufacturing can then be used for the economical production of these immunoglobulin reagents in bacterial fermentation systems, and expression vectors with hexahistidine tags can be used to simplify downstream purification. One such Fab reagent directed against botulinum neurotoxin A/B has been shown to be sensitive in a variety of assay formats including surface plasmon resonance (SPR), flow cytometry, enzyme linked immunosorbent assay (ELISA), and hand-held immunochromatographic assay. Recombinant antibodies can provide another source of high quality detection reagents in our arsenal to identify or detect pathogens in environmental samples.     

A reduction-triggered fluorescence probe for sensing nucleic acids

We have developed a reduction-triggered fluorescence probe with a new fluorogenic compound derivatized from Rhodamine for sensing oligonucleotides. The chemistry to activate the compound involves the reaction between the azide group of rhodamine derivatives and the reducing reagents, with the fluorescence signal appearing after reduction of the azide group. The signal/background ratio of this fluorogenic compound reached 2100-fold enhancement in fluorescence intensity. Dithio-1,4-threitol or triphenylphosphine as reducing reagents were successfully utilized for this chemistry to be introduced into the DNA probe. The genetic detection requires that two strands of DNA bind onto target oligonucleotides, one probe carrying a reducible fluorogenic compound while the other carries the reducing reagents. The reaction proceeds automatically without any enzymes or reagents under biological conditions to produce a fluorescence signal within 10-20 min in the presence of target DNA or RNA. In addition, the probe was very stable under biological conditions, even such extreme conditions as pH 5 solution, pH 10 solution, or high temperature (90 degrees C) with no undesirable background signal. The probes were successfully applied to the detection of oligonucleotides at the single nucleotide level in solution and endogenous RNA in bacterial cells.     

Standardization in Biological Staining. The Influence of Dye Manufacturing

The purpose of biological staining is to obtain specimens of biological material that can be assessed in the microscope. These specimens are influenced by all processes from removal from the intact organism to mounting on the microscopic slide. To achieve comparable results with various techniques for biological staining, standardization of all procedures and reagents is mandatory. In this paper, I focus particularly on dyes and consider the possibilities for obtaining standardized dyes. In general practice, most biological staining takes place with available commercial dyes. These dyes may or may not have been subjected to quality assessment either internally by the producer or vendor or externally by independent investigators or organizations such as the Biological Stain Commission. Concerted attempts at standardization in Europe are discussed. The latest results of this work, the European standard EN 12376, is presented. This standard is concerned with information supplied by the manufacturer with in vitro diagnostic reagents for biological staining. The standard has been prepared by a Working Group on Staining in Biology under Technical Committee 140, In Vitro Medical Devices, of the European committee for standardization, CEN.     

Standardization in biological staining. The influence of dye manufacturing.

The purpose of biological staining is to obtain specimens of biological material that can be assessed in the microscope. These specimens are influenced by all processes from removal from the intact organism to mounting on the microscopic slide. To achieve comparable results with various techniques for biological staining, standardization of all procedures and reagents is mandatory. In this paper, I focus particularly on dyes and consider the possibilities for obtaining standardized dyes. In general practice, most biological staining takes place with available commercial dyes. These dyes may or may not have been subjected to quality assessment either internally by the producer or vendor or externally by independent investigators or organizations such as the Biological Stain Commission. Concerted attempts at standardization in Europe are discussed. The latest results of this work, the European standard EN 12376, is presented. This standard is concerned with information supplied by the manufacturer with in vitro diagnostic reagents for biological staining. The standard has been prepared by a Working Group on Staining in Biology under Technical Committee 140, In Vitro Medical Devices, of the European committee for standardization, CEN.     

Engineered bromodomains to explore the acetylproteome

MS‐based analysis of the acetylproteome has highlighted a role for acetylation in a wide array of biological processes including gene regulation, metabolism, and cellular signaling. To date, anti‐acetyllysine antibodies have been used as the predominant affinity reagent for enrichment of acetyllysine‐containing peptides and proteins; however, these reagents suffer from high nonspecific binding and lot‐to‐lot variability. Bromodomains represent potential affinity reagents for acetylated proteins and peptides, given their natural role in recognition of acetylated sequence motifs in vivo. To evaluate their efficacy, we generated recombinant proteins representing all known yeast bromodomains. Bromodomain specificity for acetylated peptides was determined using degenerate peptide arrays, leading to the observation that different bromodomains display a wide array of binding specificities. Despite their relatively weak affinity, we demonstrate the ability of selected bromodomains to enrich acetylated peptides from a complex biological mixture prior to mass spectrometric analysis. Finally, we demonstrate a method for improving the utility of bromodomain enrichment for MS through engineering novel affinity reagents using combinatorial tandem bromodomain pairs.     

Computational detection and suppression of sequence-specific off-target phenotypes from whole genome RNAi screens

A challenge for large-scale siRNA loss-of-function studies is the biological pleiotropy resulting from multiple modes of action of siRNA reagents. A major confounding feature of these reagents is the microRNA-like translational quelling resulting from short regions of oligonucleotide complementarity to many different messenger RNAs. We developed a computational approach, deconvolution analysis of RNAi screening data, for automated quantitation of off-target effects in RNAi screening data sets. Substantial reduction of off-target rates was experimentally validated in five distinct biological screens across different genome-wide siRNA libraries. A public-access graphical-user-interface has been constructed to facilitate application of this algorithm.     

Determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays

In this paper, we report an experimental setup and mathematical algorithm for determination of relative protein abundance from directly labeled native protein samples applied to an array of antibodies. The application of the proposed experimental system compensates internally at each array element for a number of deficiencies in array experiments such as differential labeling efficiency in dual color assay systems, differential solubility of protein molecules in dual color assay systems, and differential affinity of capture reagents toward proteins labeled with two different fluorescent dyes. This system offers full compensation for variable amounts of capture reagents on separate array structures, as well as limited compensation for nonspecific interactions between capture reagents and analytes. The proposed experimental strategy enables the use of a large number of capture reagents to develop a true multiplex analysis system that will yield complete relative protein abundance information in two biological systems.     

Design and synthesis of visible isotope-coded affinity tags for the absolute quantification of specific proteins in complex mixtures

Identification of proteins in complex mixtures by mass spectrometry is most useful when quantitative data is also obtained. We recently introduced isotope-coded affinity tags (ICAT reagents) for the relative quantification of proteins present in two or more biological samples. In this report, we describe a new generation of ICAT reagents that contain the following additional features: (1) a visible tag that allows the electrophoretic position of tagged peptides during separation to be easily monitored; (2) a photocleavable linker that allows most of the tag to be removed prior to mass spectrometric analysis; (3) an isotope tag that contains carbon-13 and nitrogen-15 atoms instead of deuterium to ensure precise comigration of light and heavy tagged peptides by reverse-phase HPLC. These reagents contain an iodoacetyl group that selectively reacts with peptide cysteine residues. Peptide modification chemistry is also reported that allows tagging of peptides that are devoid of cysteine. The synthesis of these visible isotope-coded affinity tags (VICAT reagents), and their reaction with peptides are described in this report. VICAT reagents containing a carbon-14 visible probe or an NBD fluorophore are described. These reagents are most useful for the determination of the absolute quantity of specific target proteins in complex protein mixtures such as serum or cell lysates.     

Chemical modification of lactose repressor protein using N-substituted maleimides.

Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.     

Use of fluorescein hydrazide and fluorescein thiosemicarbazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized protein on polyacrylamide gels

Highly fluorescent thiosemicarbazide and hydrazide prepared by reaction of fluorescein isothiocyanate with hydrazine or adipic acid dihydrazide have been used to monitor the presence of carbonyl groups in oxidatively modified proteins. After oxidation, proteins react with these reagents under anaerobic conditions in the dark to yield fluorescent protein conjugates (presumably thiosemicarbazones or hydrazones) which can be visualized as fluorescent bands following electrophoresis (0-4 degrees C) on lithium dodecyl sulfate-polyacrylamide gels. These reagents do not react with unoxidized proteins. The conjugates formed dissociate readily at room temperature but are fairly stable at pH 6-9, 0 degrees C. Current data suggest that these reagents will be useful in the detection and quantitation of oxidatively modified proteins in biological systems.     

Electrospray mass spectra of three proprietary detergents

We have determined the major ingredients of the commercially available reagents M-PER, Y-PER, and B-PER from Pierce Chemical Co. using electrospray mass spectrometry. These three proprietary reagents have been widely used in the biochemical community as cell membrane dissolving tools during the initial step of protein purification. However, the identity and mechanism of these reagents remained unknown. In this paper, we identified these reagents as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and n-octyl-beta-d-thioglucopyranoside, respectively. In addition, we wish to stress here the increasing importance of the role of electrospray mass spectrometry in the analysis of such proprietary biological preparations which are increasingly finding their way into the biochemical literature.     

The oxidative cleavability of protein cross-linking reagents containing organoselenium bridges

The intensive use of cleavable cross-linking reagents to study macromolecular biological interactions has shown a demand for optimizing these reagents in such a way that the involved macromolecules remain intact. The present work focuses on the development of selenium linkers that are cleavable by mild oxidation. The efficiency of cross-linking and subsequent cross-linker cleavage with a new series of such homo- or heterobifunctional cross-linking reagents have been tested in a simple model system, consisting of albumin and cytochrome c. Resultant, or residual, covalent complex formation is examined by SDS-polyacrylamide gel electrophoresis. From this work it can be concluded that diallyl selenides are readily cleaved by mild oxidation, whereas dialkyl selenides and benzyl alkyl selenides can only be cleaved when the alkyl part of the selenide has an electron-withdrawing group next to the beta-carbon from selenium.     

Kits and their unique role in molecular biology: a brief retrospective

Since their initial development nearly 20 years ago, molecular biology kits have evolved from simple protocols and reagents for cloning of DNA to the more recent complex reagent sets that enable whole genomic sequencing. Initially met with resistance by some who felt that using them deprived researchers of the basic knowledge of how to create reagents, molecular biology kits have taken on an important role in the biological sciences. In this article we describe kit development, why kits have succeeded in molecular biology, and how they have paved the way for the more recent widespread use of core facilities.     

New reagents, reactions, and peptidomimetics for drug design.

It has been a major focus in our laboratories to prepare novel reagents and peptidomimetic structures for drug design. We have designed and prepared novel guanidinylation reagents that can be employed in solution or as solid phase reagents. We and others have utilized the reagent 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) for amide bond formation to couple sterically hindered structures. These couplings proceed with remarkably strong resistance to racemization. In the area of peptidomimetics, we have incorporated novel building blocks to create biologically active compounds. These building blocks include thioether and alkylamine bridges, beta-methylated, and beta,beta-dimethylated amino acid residues. These mimetic structures have been incorporated into specific target molecules such as opioids to obtain cyclic peptidomimetics with potent and selective biological activity.