Nanostructured dna-protein aggregates consisting of covalent oligonucleotide-streptavidin conjugates

Covalent conjugates consisting of streptavidin and a 24-mer single-stranded DNA oligonucleotide have been oligomerized by cross-linking with a 5',5'-bis-biotinylated 169-base-pair double-stranded DNA (dsDNA) fragment. The oligomeric conjugates formed have been analyzed by nondenaturing gel electrophoresis and scanning-force microscopy (SFM). The comparison of analogous oligomers, prepared from native STV and the bis-biotinylated dsDNA fragment, revealed that the covalent STV-oligonucleotide hybrid conjugates self-assemble to generate oligomeric aggregates of significant smaller size, containing on average only about 2.5 times less dsDNA fragments per aggregate. Likely, this is a consequence of electrostatic or steric repulsion between the dsDNA and the single-stranded oligomer covalently attached to the hybrid, as indicated from control experiments. Nevertheless, the single-stranded oligonucleotide moiety within the oligomeric conjugates can be used as a selective molecular handle for further functionalization and manipulation. For instance, it was used for specific DNA-directed immobilization at a surface, previously functionalized with complementary capture oligonucleotides. Moreover, we demonstrate that macromolecules, such as STV and antibody molecules, which are tagged with the complementary oligonucleotide, specifically bind to the supramolecular DNA-STV oligomeric conjugates. This leads to a novel class of functional DNA-protein conjugates, suitable, for instance, as reagents in immuno-PCR or as building blocks in molecular nanotechnology.     

Combination of DNA-directed immobilization and immuno-PCR: very sensitive antigen detection by means of self-assembled DNA-protein conjugates

An assay for very sensitive antigen detection is described which takes advantage of the self- assembly capabilities of semi-synthetic conjugates of DNA and proteins. The general scheme of this assay is similar to a two-sided (sandwich) enzyme-linked immunoassay (ELISA); however, covalent single-stranded DNA-streptavidin (STV) conjugates, capable of hybridizing to complementary surface-bound DNA oligomers, are utilized for the effective immobilization of either capture antibodies or antigens, rather than the chemi- or physisorption usually applied in ELISA. Immuno-PCR (IPCR) is employed as a method for signal generation, utilizing oligomeric reagents obtained by self-assembly of STV, biotinylated DNA and antibodies. In three different model systems, detecting human IgG, rabbit IgG or carcinoembryonic antigen, this combination allowed one to increase the sensitivity of the analogous ELISA approximately 1000-fold. For example, <0.1 amol/ micro l (15 pg/ml) of rabbit IgG was detectable. The immunoassay can be carried out in a single step by tagging the analyte with both reagents for capture and read-out simultaneously, thereby significantly reducing handling time and costs of analysis. Moreover, as the spatial selectivity of target immobilization is determined by the specificity of DNA base pairing, the assay is particularly suited for miniaturized microfluidics and lab-on-a-chip devices.     

Combination of DNA-directed immobilization and immuno-PCR: very sensitive antigen detection by means of self-assembled DNA–protein conjugates

An assay for very sensitive antigen detection is described which takes advantage of the self- assembly capabilities of semi-synthetic conjugates of DNA and proteins. The general scheme of this assay is similar to a two-sided (sandwich) enzyme-linked immunoassay (ELISA); however, covalent single-stranded DNA–streptavidin (STV) conjugates, capable of hybridizing to complementary surface-bound DNA oligomers, are utilized for the effective immobilzation of either capture antibodies or antigens, rather than the chemi- or physisorption usually applied in ELISA. Immuno-PCR (IPCR) is employed as a method for signal generation, utilizing oligomeric reagents obtained by self-assembly of STV, biotinylated DNA and antibodies. In three different model systems, detecting human IgG, rabbit IgG or carcinoembryonic antigen, this combination allowed one to increase the sensitivity of the analogous ELISA ~1000-fold. For example, <0.1 amol/µl (15 pg/ml) of rabbit IgG was detectable. The immunoassay can be carried out in a single step by tagging the analyte with both reagents for capture and read-out simultaneously, thereby significantly reducing handling time and costs of analysis. Moreover, as the spatial selectivity of target immobilization is determined by the specificity of DNA base pairing, the assay is particularly suited for miniaturized microfluidics and lab-on-a-chip devices.     

Supramolecular DNA-streptavidin nanocircles with a covalently attached oligonucleotide moiety

Covalent hybrid conjugates consisting of streptavidin (STV) and a 24-mer single-stranded DNA oligonucleotide have been used as a starting material for the synthesis of supramolecular nanocircles. For this, the covalent hybrid conjugates were oligomerized by cross-linking with 5 ,5 -bis-biotinylated double-stranded DNA (dsDNA) fragments of various length. Heat denaturation of the resulting oligomeric conjugates and subsequent rapid cooling led to the formation of the nanocircles, in which the oligonucleotide-containing STV molecule is coupled with both ends of the circular bis-biotinylated dsDNA fragment. The circular structure of the bioconjugates was established by electrophoretic studies including Ferguson plot analysis as well as by scanning force microscopy (SFM) inspection. The formation process and the stability against degradation by ligand exchange with free D-biotin was compared for the nanocircles obtained from covalent oligonucleotide-STV hybrids and native STV. The former nanocircles revealed a decreased stability with respect to ring opening than the circles obtained from native STV. This suggested that the affinity of the covalent oligonucleotide-STV hybrid for binding biotinylated DNA is significantly decreased. Nevertheless, the single-stranded oligonucleotide moiety of the hybrid nanocircles can be used as a molecular handle for further functionalization. For instance, it was used for the selective DNA-directed immobilization at a surface, previously functionalized with complementary capture oligonucleotides. Moreover, we demonstrate that a pair of nanocircles, containing complementary oligonucleotide moieties, can be hybridized to form specific dimers, thereby generating a novel type of supramolecular DNA-protein nanostructures.     

Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidin-biotin attachment: a comparative study

Antibody microarrays have the potential to revolutionize protein diagnostics. The major problems in the fabrication of antibody arrays, however, concern the reproducibility and homogeneity of the attachment of the proteins on the solid substrate. We here compare the DNA-directed immobilization (DDI) method with two conventional strategies for immobilization of antibodies on glass substrates. DDI is based on the self-assembly of semisynthetic DNA-streptavidin conjugates which converts an array of DNA oligomers into an antibody microarray. DDI was compared with direct spotting of antibodies on chemically activated glass slides and with immobilization of biotinylated antibodies on streptavidin-coated slides. The immobilized antibodies were used as capture reagents in a two-sided (sandwich) immunoassay for the quantification of rabbit IgG as a model antigen. Detection limits down to 0.001nM (150 pg/mL) were attained with all three array formats; however, DDI and direct spotting of the antibodies led to the highest fluorescence intensities. DDI led to the best spot homogeneity and intra- and interexperimental reproducibility. Moreover, DDI allowed highly economical use of antibody materials; that is, at least 100-fold less antibody is needed for preparing an array by DDI instead of by direct spotting. Taking into account the greater versatility and convenience of handling of the self-assembly approach, this study demonstrates that DDI is an advantageous alternative for generating versatile and robust protein arrays.     

A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins

A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA-protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1-0.01 amol (500-50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive.     

Applications of real-time immuno-polymerase chain reaction (rt-IPCR) for the rapid diagnoses of viral antigens and pathologic proteins

One of the major challenges of performing the IPCR has been to establish a robust, sensitive, and specific method which is easily adapted and highly standardized for routine use in a clinical laboratory. Presently, the performance of IPCR typically involves elaborate and multiple, time-consuming steps prone to high variation in reagents and technical application. Further advances in the technology and instrumentation used for the signal detection of IPCR has resulted in the development of real-time IPCR (rt-IPCR). Rt-IPCR is still relatively undeveloped in comparison to the use of both real-time PCR and IPCR as evidenced by the low number (eight citations) of publications in the scientific literature. However, increased use of rt-IPCR has shown that the method displays improved statistical validation of accuracy over IPCR. Inter-assay error is typically 5-10% vs 15-20% for IPCR. The primary advantage of using rt-IPCR in place of IPCR is the immediate interpretation of positive data (quantification of proteins) as the PCR reaction proceeds. This aspect is key to real-time diagnosis and has great importance for specific emergency situations (i.e., biological and environmental contaminations of toxins in biothreat situations), as well as cases where specific tumor/viral antigens and pathologic proteins may be present in body tissues in extremely low concentrations and rapid, early diagnosis is important for immediate palliative treatment. This review summarizes all of the experimental data published to date utilizing the rt-IPCR method for various analytes (vascular endothelial growth factor, mumps Ag, rViscumin, various IgG, gliadin, HIV-1 p24 Ag, Rotavirus VP6, pathologic and recombinant prion, and prostate specific Ag) and describes the molecular scaffold formats, solid formats, instrument detection systems, and probes/primers or fluorescent dyes used in these assays. With further standardization and validation, rt-IPCR has the potential to become the most analytically sensitive method available for the detection of proteins.     

95 Orientational ordering of short DNA fragments in the polymer matrix

As a vital part of modern nanotechnology, nanofabrication aims to develop nanoscale components and nanomaterials in large quantities at relatively low cost. The promising strategy is the bottom-up self-assembly techniques of chemical assembly and molecular recognition to bring together individual atoms, molecules, or supramolecular building blocks to form useful constructs. The DNA-DNA self-assembly seems to be the key point regulating the polymer composites formation. We address the mixture of a flexible polymer with short double-strand DNA fragments, where the persistence length is in comparable with the contour length of the molecule. We investigate the conditions affecting the orientational order formation of short double-strand DNA fragments, immersed in the flexible polymer. It is shown that short double-strand DNA fragments exhibit the formation of a liquid crystalline ordered phase, in dependence on the value of the Flory–Huggins parameter, aspect ratio , and the attraction energy (Mamasakhlisov et al., 2009; Todd et al., 2008) of the double strand DNA molecules and volume fraction of polymer.     

中国地鼠基因组微卫星富集文库的构建与分析

目的筛选中国地鼠微卫星位点,为中国地鼠种质资源的分类、进化等遗传研究奠定基础。方法中国地鼠基因组DNA经超声打碎,用2%琼脂糖凝胶电泳回收500～1000 bp的DNA片段,与SNX连接头连接,连接产物与生物素标记的14种微卫星探针变性及退火,再通过链亲和素偶联磁珠亲和捕捉,经吸附、洗涤及洗脱,然后以洗脱产物为模板,通过PCR扩增,与pGEM-T载体连接,转化大肠杆菌DH10B,构建中国地鼠微卫星DNA富集文库。结果测序结果发现,微卫星DNA序列的阳性克隆占70.3%。结论中国地鼠微卫星文库的建立和微卫星的筛选将为下一步进行中国地鼠遗传连锁图谱的构建、分子进化和系统发育研究提供大量的微卫星标记。     

Evaluation of single-stranded nucleic acids as carriers in the DNA-directed assembly of macromolecules

Current developments in nanosciences indicate that the self-assembly of macromolecules, such as proteins or metallic nanoclusters, can be conveniently achieved by means of nucleic acid hybridization. Within this context, we here report on the evaluation of single-stranded nucleic acids to be utilized as carrier backbones in DNA-directed self-assembly. A microplate solid-phase hybridization assay is described which allows rapid experimental determination of the hybridization efficiencies of various sequence stretches within a given nucleic acid carrier strand. As demonstrated for two DNA fragments of different sequence, the binding efficiencies of several oligonucleotides depend on the formation of specific secondary structure elements within the carrier molecule. A correlation of sequence-specific hybridization capability with modeled secondary structure is also obvious from experiments using the fluorescence gel-shift analysis. Electrophoretic studies on the employment of helper oligonucleotides in the formation of supramolecular conjugates of several oligonucleotide-tagged proteins indicate, that structural constraints can be minimized by disruption of intramolecular secondary structures of the carrier molecule. To estimate the influences of the chemical nature of the carrier, gel-shift experiments are carried out to compare a 170mer RNA molecule with its DNA analogue. Ternary aggregates, containing two protein components bound to the carrier, are formed with a greater efficiency on the DNA instead of the RNA carrier backbone.     

The developments of semisynthetic DNA-protein conjugates

Semisynthetic DNA-protein conjugates, generated by either covalent or non-covalent coupling chemistry, are versatile molecular tools applicable in bioanalytical and synthetic chemical procedures. This article reviews the synthesis and characterization of artificial nucleic acid-protein conjugates, in addition to applications arising in the life sciences and nanobiotechnology, such as the self-assembly of high-affinity reagents for immunological detection assays and biosensors, the fabrication of laterally microstructured biochips, and the biomimetic 'bottom-up' synthesis of nanostructured supramolecular devices.     

利用Inverse PCR快速筛选单个T-DNA拷贝转基因水稻植株的方法

为了获得单个T-DNA插入拷贝的植株, 我们建立了一套利用Inverse PCR(IPCR)快速检测转基因水稻中T-DNA拷贝数的方法。用IPCR的方法可以扩增出与已知T-DNA序列相邻的水稻基因组DNA未知序列,由此推测转基因水稻植株中T-DNA的拷贝数。我们共对15个转化株系20棵不同植株的DNA进行了IPCR检测。其中12株表现为T-DNA单拷贝插入,3株为双拷贝插入,1株为三拷贝插入。另外4株未检测到T-DNA插入拷贝。IPCR分析结果经过Southern杂交和测序的验证。     

DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3′-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.     

DNA sequencing with solid-phase-capturable dideoxynucleotides and energy transfer primers

False terminations occurring in fluorescent dye-primer DNA sequencing, and nonsequencing primer extension DNA fragments generated in dye-terminator sequencing cause background noise in fluorescent electropherograms, leading to errors in sequence determination. We describe here a DNA sequencing chemistry that produces accurate and clean sequencing data on a fluorescent DNA sequencer, eliminating the false terminations and background noise. The procedure involves coupling fluorescence energy transfer (ET) primers that produce high fluorescent signals with solid-phase-capturable biotinylated dideoxynucleotides to generate Sanger DNA sequencing fragments. After the sequencing reaction,the DNA extension fragments that carry a biotin at the 3' end are captured with streptavidin-coated magnetic beads, while the other components in the sequencing reaction are washed away. Only pure DNA extension products terminated by the biotinylated dideoxynucleotides are released from the magnetic beads and are loaded onto a sequencing gel to produce accurate sequencing data.     

Enhanced resolution of random amplified polymorphic DNA genotyping of Pseudomonas aeruginosa

AIMS: To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa. METHODS AND RESULTS: Ps. aeruginosa genomic DNA extraction, RAPD-PCR, electrophoresis on acrylamide gel and silver staining were performed by using standardized reagents and conditions. The results were compared with the agarose gel electrophoresis followed by ethidium bromide staining. CONCLUSIONS: The coupling of acrylamide gel electrophoresis and silver staining gave about 80% more DNA bands than the traditional method, allowing a finer discrimination among different Ps. aeruginosa strains. SIGNIFICANCE AND IMPACT OF THE STUDY: By enhancing the resolution of the electrophoretic separation and the sensitivity of the staining, random amplification could be easily applied to the surveillance and prevention of nosocomial infections by clinical microbiology laboratories.     

Measuring single-bond rupture forces using high electric fields in microfluidic channels and DNA oligomers as force tags

The disruption force of specific biotin-streptavidin bonds was determined using DNA oligomers as force tags. Forces were generated by an electric field acting on a biotinylated fluorescently labeled DNA oligomer. DNA oligomers were immobilized via biotin-streptavidin bonds on the walls of microfluidic channels. Channel layout and fluid-based deposition process were designed to enable well-defined localized deposition of the oligomers in a narrow gap of the microchannel. Electric fields of up to 400 V/cm were applied and electric field induced desorption of DNA oligomers was observed. At T approximately 30 degrees C, field-induced desorption of both a 12 mer as well as a 48 mer yielded a streptavidin-biotin disruption force of 75 fN. Streptavidin-functionalized surfaces remained intact and could be reloaded with biotinylated oligomers. At approximately 20 degrees C, however, no field-induced unbinding of the oligomers was observed at electric field strength of up to 400 V/cm, indicating a significant temperature dependence of the bond strength.     

DNA fragmentation pattern induced in thymocytes by sulphur mustard

Sulphur mustard (HD) is a blister agent for which no specific therapy exists. The mechanism of cell injury caused by HD is not well understood. This study examined DNA damage in thymocytes exposed to a range of HD concentrations over a time course of 1-24 h. Thymocytes incubated with HD showed an increase in the production of DNA fragments of the type frequently associated with apoptosis, namely, initial formation of large fragments of 30-50, 200-300 and > 700 kilobase pairs (kbp), followed by further degradation to produce an internucleosomal 'ladder' of oligomers of approximately 180 base pairs (bp). Pulsed field electrophoresis analysis of thymocytes incubated with HD detected breakdown of the chromatin up to 3 h before a corresponding increase in the low molecular weight (MW) oligonucleosomal fragments could be seen on conventional agarose gels. These results suggest that cells damaged by HD poisoning may be irretrievably committed to cell death sooner after exposure than previous studies suggested. The nature of the DNA fragments produced suggested that apoptosis may represent a component of the pathway of cell death induced by HD. These aspects may have implications for the search for specific therapeutic reagents effective in the prevention or treatment of HD poisoning.     

Short Oligonucleotide Tandem Ligation Assay for Genotyping of Single-Nucleotide Polymorphisms in Y Chromosome

We propose a novel universal methodology, Short Oligonucleotide Tandem Ligation Assay (SOTLA), for SNP genotyping. SOTLA is based on using a tandem of short oligonucleotide (TSO) probes consisting of three fragments: the core oligonucleotide and two flanking oligomers, one of which is immobilized onto a solid support and another one contains the biotin label. TSO is self-associated on a complementary DNA template, forms the complex containing two nicks, which are efficiently ligated with DNA ligase giving biotinylated oligonucleotide covalently bound to polymer beads. No ligation of TSO on an imperfect DNA template bearing the base substitution in the core binding site is occurred. We used SOTLA for the highly selective SNP analysis in different DNA fragments of human Y chromosome. Comparison of SOTLA results with those of PCR-RFLP and allele-specific PCR techniques demonstrates that SOTLA ensures the univocal reliable SNP analysis in different PCR fragments varying in length and base composition. The fundamental difference between SOTLA and well known OLA approaches while using T4 DNA ligase is that the accuracy of SNP analysis in OLA is ensured only by the specificity of ligase while that in SOTLA is provided by the specificity of both ligation and hybridization of TSO probes.     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.