丙型肝炎病毒诊断参考品的研究进展

体外诊断试剂的参考品是由逐一反复论证的一套样品所组成,用于全面控制试剂质量,它对于试剂应达到的各种指标提出了全面要求。目前国际上常用的丙型肝炎诊断试剂参考品为抗HCV抗体血清参考品和核酸检测参考品,我们简要综述了丙型肝炎病毒诊断试剂参考品的研制及发展。     

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.     

Studies towards the development of chemically synthesized non-radioactive biotinylated nucleic acid hybridization probes.

Non-radioactive nucleic acid hybridization probes have been constructed in which the reporter group is long chain biotin chemically linked to a basic macromolecule (histone H1, cytochrome C or polyethyleneimine). The modified basic macromolecule which carries many biotin residues can, in turn, be covalently linked to nucleic acids (DNA) via the bifunctional cross-linking reagents, glutaraldehyde, 1,2,7,8-diepoxyoctane, bis (succinimidyl) suberate or bis (sulfonosuccinimidyl) suberate. This provides a very sensitive probe by which as little as between 10-50fg of target DNA can be visualized using dot-blot hybridization procedures in conjunction with avidin or streptavidin enzyme conjugates.     

Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system

A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol buffer solutions for dissolving freeze-dried buffer components, rinsing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, heats, mixes reagents, and flows solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.     

Reagents specific for cell surface components.

Mercury, diazonium ions and dyes which bind nucleic acids were covalently linked to dextrans using methods that resulted in non-hydrolyzable reagent-dextran bonds without impairing the binding abilities of the reagents, i.e. these dextran derivatives reacted with thiols, phenols/imidazoles and nucleic acids respectively. Since these dextran derivatives cannot penetrate into cells and since dextran itself does not bind to cells, these compounds represent reagents specific for the cell surface. They may be used both to evaluate cell surface constituents of intact cells and to affect viable cells via an interaction with those constituents. Mercury-dextran was found to bind to cells; the amount of mercury thus attached to the cells was about ten times smaller than when an equivalent concentration of free mercury ions was used. Mercury-dextran, bound to cells after a 30-min exposure at room temperature, was localized on the surface of these cells, as sodium borohydride reduced this complex giving rise to the intact cells, elementary mercury and free dextran which was released into medium. When cells were constantly exposed to the mercury-dextran, its toxic effects were comparable to that of free mercury ions. Diazonium-dextran, which also binds tightly to the cell surface, was also considerably toxic. Dextrans substituted with dyes which bind to nucleic acids were less toxic than the parent dyes themselves; it was shown that the attachment of such a dye to dextran decreased the binding of dye to cells under detection limits.     

Identification of thyroid hormone receptors in rat liver nuclei by photoaffinity labeling with L-thyroxine and triiodo-L-thyronine

Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoduplication and fluorescence measurement in polyacrylamide gel electrophoresis.

A simple method of photoduplication of gel electrophoresis, visualized with fluorescent reagents, is described. The procedure is convenient and rapid and requires no camera or expensive equipment. Using electrophoresis duplicating paper (Kodak), positive prints suitable for documentation or publication may be obtained. With usual photographic paper, negative prints may be obtained, allowing reliable measurement by scanning. The technique may be applied to protein or nucleic acid electrophoresis.     

A sense of closeness: protein detection by proximity ligation

Highly specific and sensitive procedures will be required to evaluate proteomes. Proximity ligation is a recently introduced mechanism for protein analysis. In this technique, the convergence of sets of protein-binding reagents on individual target molecules juxtaposes attached nucleic acid sequences. Through a ligation reaction a DNA reporter sequence is created, which can be amplified. The procedure thus encodes detected proteins as specific nucleic acid sequences in what may be viewed as a reverse translation reaction.     

Polyfunctional molecules and their components in the processes of aromatic nucleophilic substitution. II. Nucleophilic modification of 3',5'-bis-O-(alpha,beta,alpha',beta'-tetrafluoropyridyl-gamma)thymidine

The interaction of 3',5'-bis-O-(alpha,beta,alpha',beta'-tetrafluoropyrid-gamma-yl)thymidine with various nucleophilic reagents was studied to evaluate the possibility of molecular design of new types of nucleic acid analogues using SNAr reactions. The reactions with morpholine and sodium azide led to the introduction of one and two nucleophilic residues into each of the polyfluorinated pyridine rings. The nucleophilic polycondensation with bifunctional reagents ethylenediamine and hexamethylenediamine depended on the nature of nucleophile and reaction conditions and resulted in the formation of supramolecules containing about five or more than 20 pyrimidine bases.     

Photoaffinity modification of delta 5-3-ketosteroid isomerase by light-activatable steroid ketones covalently coupled to agarose beads

In order to identify the minor site(s) of photoattachment of unsaturated steroid ketones to delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni, we have developed a solid-state photoaffinity labeling technique. Two solid-state reagents, O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-19-nortestosterone and O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadien-3-one, have been synthesized. Under anaerobic conditions, isomerase bound to these resins is photoinactivated by UV light (lambda greater than 290 nm) whereas isomerase bound to O-carboxymethylagarose-ethylenediamine-deoxycholate or isomerase in the presence of O-carboxymethylagarose-ethylenediamine-acetate is almost completely stable to irradiation under the same conditions. Photoinactivation under anaerobic condition promoted by the resin-bound steroid ketones results from a reaction at the active site since the competitive inhibitor, sodium cholate, which does not absorb light above 290 nm, provides protection toward photoinactivation. Preliminary analysis of isomerase that has been photolyzed in the presence of O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadiene-3-one has established that the enzyme is converted to at least two different forms. One form binds more tightly to the resin than does the native enzyme. This form can be eluted by a sodium dodecyl sulfate containing buffer. The second form is not eluted by this buffer but can be released from the resin by cleavage of the ester bond linking the steroid to the derivatized agarose. We presume that the latter form is covalently coupled to the resin-linked steroid. In the presence of oxygen, additional nonspecific inactivation reactions occur, but these can be suppressed by the singlet oxygen trap, L-histidine. The application of solid-state photoaffinity reagents to some areas of receptor isolation and characterization is discussed.     

Semi-synthetic nucleic acid-protein conjugates: applications in life sciences and nanobiotechnology

Semi-synthetic conjugates of nucleic acids and proteins can be generated by either covalent coupling chemistry, or else by non-covalent biomolecular recognition systems, such as receptor-ligands of complementary nucleic acids. These nucleic acid-protein conjugates are versatile molecular tools which can be applied, for instance, in the self-assembly of high-affinity reagents for immunological detection assays, the fabrication of laterally microstructured biochips containing functional biological groups, and the biomimetic 'bottom-up' synthesis of nanostructured supramolecular devices. This review summarizes the current state-of-the-art synthesis and characterization methods of artificial nucleic acid-protein conjugates, as well as applications and perspectives for future developments of such hybrid biomolecular components in life sciences and nanobiotechnology.     

Reactive oligonucleotide derivatives as gene-targeted biologically active compounds and affinity probes

Development of efficient methods for synthesis of oligonucleotides and oligonucleotide analogs has opened up the possibility of designing a broad spectrum of affinity reagents for specific modification of nucleic acids and proteins. These affinity reagents are used for investigation of the topology of ribosomes and nucleic acid polymerases. Oligonucleotides and their analogs are already used for suppression of specific gene expression and for elucidation of the physiological role of their products. Oligonucleotide derivatives appear to offer considerable promise as potential gene-targeted drugs such as antivirals and specific inhibitors of oncogene expression.     

Intracellular delivery of nucleic acid by cell-permeable hPP10 peptide

Although gene therapy offers hope against incurable diseases, nonreplicating transduction vectors remain lacking. We have previously characterized a cell-penetrating peptide hPP10 for the delivery of various cargoes; however, whether hPP10 can mediate nucleic acid delivery is still unknown. Here, examining via different ways, we demonstrate that hPP10 stably complexes with plasmid DNA (pDNA) and safely mediates nucleic acid transfection. hPP10 can mediate GFP-, dsRed-, and luciferase-expressing plasmids into cells with nearly the same efficiency as commercial transfection reagents Turbofectin or Lipofect. Furthermore, hPP10 can mediate Cre fusion protein delivery and pDNA transfection simultaneously in the Cre/loxp system in vitro. In addition, hPP10 fused with an RNA-binding domain can mediate delivery of small interfering RNA into cells to silence the reporter gene expression. Collectively, our results suggest that hPP10 is an option for nucleic acid delivery with efficiencies similar to that of commercial reagents.     

In vitro selection of high-affinity nucleic acid ligands to parasite target molecules

The logic of using nucleic acids as pharmaceutical reagents is in part based on their capacity to interact with high affinity and specificity with other biological components. Considerable progress has been made over the past 10 years in the development of nucleic acid-based drug molecules using a variety of different technologies. One approach is a combinatorial technology that involves an iterative Darwinian-type in vitro evolution process, which has been termed SELEX for 'systematic evolution of ligands by exponential enrichment'. The procedure is a highly efficient method of identifying rare ligands from combinatorial nucleic acid libraries of very high complexity. It allows the selection of nucleic acid molecules with desired functions and it has been instrumental in the identification of a number of synthetic DNA and RNA molecules, so-called aptamers that recognise ligands of different chemical origin. The method is fast, it does not require special equipment and the selected aptamers typically bind their target with high affinity and high specificity. Here we summarise the recent examples of the SELEX technique within the context of identifying high-affinity ligands against parasite target molecules.     

Escherichia coli RNase D. Purification and structural characterization of a putative processing nuclease

RNase D, a putative tRNa processing nuclease, has been purified about 1,000-fold from extracts of Escherichia coli to apparent homogeneity, as judged by acrylamide gel electrophoresis under nondenaturing and denaturing conditions and by gel electrofocusing. The purified enzyme is a single chain protein with a molecular weight of 40,000 and an isoelectric point of about 6.2. Spectral analysis indicated that RNase D is devoid of nucleic acid. Amino acid analysis suggested a low content of cysteine, and this was confirmed by the relative insensitivity of the enzyme to sulfhydryl group reagents. RNase D is sensitive to inactivation by elevated temperatures but can be protected by a variety of RNAs, including those which are not substrates for hydrolysis. The relation of RNase D to other known E. coli ribonucleases and to other previously identified processing activities, is discussed.     

In vitro selection of functional nucleic acid sequences

The power of in vitro selection methods for the isolation of nucleic acids that display a desired property derives from the enormous number of sequence variants that can be surveyed with relative ease using controlled in vitro biochemistry. This methodology has found a variety of applications, ranging from the study of nucleic acid-protein interactions and natural ribozymes to the isolation of nucleic acids with potential as diagnostic or therapeutic reagents or with new catalytic activities. The number of reported applications is growing exponentially, and each application presents new variables and challenges. The goal of this article is to guide prospective users through the myriad decisions that must be made in the design and execution of a successful in vitro selection experiment.     

A guide to simple and informative binding assays

The aim of binding assays is to measure interactions between two molecules, such as a protein binding another protein, a small molecule, or a nucleic acid. Hard work is required to prepare reagents, but flaws in the design of many binding experiments limit the information obtained. In particular many experiments fail to measure the affinity of the reactants for each other. This essay describes simple methods to get the most out of valuable reagents in binding experiments.     

Standardization: a Progress Report

The introduction of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products requires the availability of well-characterized reference reagents. Working reagents for hepatitis C virus RNA, hepatitis B virus DNA, HIV-1 RNA and human parvovirus B19 DNA have been established at NIBSC and at many other laboratories (both official medicinal control laboratories and commercial laboratories). However, as these reagents have been characterised independently, it is difficult to compare results from assays using different working reagents. Recently, a WHO International Standard was established for HCV RNA NAT assays. This standard has been calibrated in International Units (IU) and provides a common standard against which all working reagents can be calibrated. Collaborative studies to characterise two further candidate International Standards for HBV DNA and HIV-1 RNA NAT assays have been completed.