Cell membrane array fabrication and assay technology

Background  

Microarray technology has been used extensively over the past 10 years for assessing gene expression, and has facilitated precise genetic profiling of everything from tumors to small molecule drugs. By contrast, arraying cell membranes in a manner which preserves their ability to mediate biochemical processes has been considerably more difficult.     

Reattachment of surface array proteins to Campylobacter fetus cells.

Campylobacter fetus strains may be of serotype A or B, a property associated with lipopolysaccharide (LPS) structure. Wild-type C. fetus strains contain surface array proteins (S-layer proteins) that may be extracted in water and that are critical for virulence. To explore the relationship of S-layer proteins to other surface components, we reattached S-layer proteins onto S- template cells generated by spontaneous mutation or by serial extractions of S+ cells with water. Reattachment occurred in the presence of divalent (Ba2+, Ca2+, Co2+, and Mg2+) but not monovalent (H+, NH4+, Na+, K+) or trivalent (Fe3+) cations. The 98-, 125-, 127-, and 149-kDa S-layer proteins isolated from strains containing type A LPS (type A S-layer protein) all reattached to S- template cells containing type A LPS (type A cells) but not to type B cells. The 98-kDa type B S-layer protein reattached to SAP- type B cells but not to type A cells. Recombinant 98-kDa type A S-layer protein and its truncated amino-terminal 65- and 50-kDa segments expressed in Escherichia coli retained the full and specific determinants for attachment. S-layer protein and purified homologous but not heterologous LPS in the presence of calcium produced insoluble complexes. By quantitative enzyme-linked immunosorbent assay, the S-layer protein copy number per C. fetus cell was determined to be approximately 10(5). In conclusion, C. fetus cells are encapsulated by a large number of S-layer protein molecules which may be specifically attached through the N-terminal half of the molecule to LPS in the presence of divalent cations.     

Modelling repressor proteins docking to DNA

The docking of repressor proteins to DNA starting from the unbound protein and model-built DNA coordinates is modeled computationally. The approach was evaluated on eight repressor/DNA complexes that employed different modes for protein/ DNA recognition. The global search is based on a protein-protein docking algorithm that evaluates shape and electrostatic complementarity, which was modified to consider the importance of electrostatic features in DNA-protein recognition. Complexes were then ranked by an empirical score for the observed amino acid /nucleotide pairings (i.e., protein-DNA pair potentials) derived from a database of 20 protein/DNA complexes. A good prediction had at least 65% of the correct contacts modeled. This approach was able to identify a good solution at rank four or better for three out of the eight complexes. Predicted complexes were filtered by a distance constraint based on experimental data defining the DNA footprint. This improved coverage to four out of eight complexes having a good model at rank four or better. The additional use of amino acid mutagenesis and phylogenetic data defining residues on the repressor resulted in between 2 and 27 models that would have to be examined to find a good solution for seven of the eight test systems. This study shows that starting with unbound coordinates one can predict three-dimensional models for protein/DNA complexes that do not involve gross conformational changes on association. Proteins 33:535–549, 1998. © 1998 Wiley-Liss, Inc.     

Growth factor array fabrication using a color ink jet printer

We have developed a novel method for growth factor analysis using a commercial color ink jet printer to fabricate substrata patterned with growth factors. We prepared substrata with insulin printed in a simple pattern or containing multiple areas of varying quantities of printed insulin. When we cultured the mouse myoblast cell line, C2C12, on the insulin-patterned substrata, the cells were grown in the same pattern with the insulin-printed pattern. Cell culture with the latter substrata demonstrated that quantity control of insulin deposition by a color ink jet printer is possible. For further applications, we developed substrata with insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) spotted in 16 different areas in varying combinations and concentrations (growth factor array). With this growth factor array, C2C12 cells were cultured, and the onset of muscle cell differentiation was monitored for the expression of the myogenic regulator myogenin. The ratio of cells expressing myogenin varied with the doses of IGF-I and bFGF in the sections, demonstrating a feasibility of growth factor array fabrication by a color ink jet printer. Since a printer manipulates several colors, this method can be easily applied to multivariate analyses of growth factors and attachment factors affecting cell growth and differentiation. This method may provide a powerful tool for cell biology and tissue engineering, especially for stem cell research in investigating unknown conditions for differentiation.     

新型的DNA金属化制作工艺

生物与微细加工技术的结合日益成为一种新趋势,DNA分子具有热力学上的稳定性、线性的分子结构及机械刚性等优点,可以作为制备纳米线的理想模板。通过DNA为模板的金属化,形成导电性好的金属纳米线和金属团簇的纳米结构,使得DNA分子作为纳米导线构筑纳米器件成为可能。在本文中,我们对几种具有代表性的DNA金属化工艺的原理进行了讨论,研究了其新型的制作工艺,通过进一步的自组装和自识别技术,金属化过的DNA就可以被用来构建电路,并作为后续的金属沉积模板,在构筑生物纳米器件的领域将有广阔的应用前景。     

Quantitative analysis of DNA array autoradiographs

DNA arrays and chips are powerful new tools for gene expression profiling. Current arrays contain hundreds or thousands of probes and large scale sequencing and screening projects will likely lead to the creation of global genomic arrays. DNA arrays and chips will be key in understanding how genes respond to specific changes of environment and will also greatly assist in drug discovery and molecular diagnostics. To facilitate widespread realization of the quantitative potential of this approach, we have designed procedures and software which facilitate analysis of autoradiography films with accuracy comparable to phosphorimaging devices. Algorithms designed for analysis of DNA array autoradiographs incorporate 3-D peak fitting of features on films and estimation of local backgrounds. This software has a flexible grid geometry and can be applied to different types of DNA arrays, including custom arrays.     

Identification of proteins bound to a thioaptamer probe on a proteomics array

A rapid method to screen and identify unknown bound proteins to specific nucleic acid probes anchored on ProteinChip array surfaces from crude biological samples has been developed in this paper. It was demonstrated with screening specific binding proteins from LPS-stimulated mouse 70Z/3 pre-B cell nuclear extracts by direct coupling of thioaptamer XBY-S2 to the pre-activated ProteinChip array surfaces. With pre-fractionation of crude nuclear extracts by ion exchange method, specific "on-chip" captured proteins have been obtained that were pure enough to do "on-chip" digestion and the subsequent identification of the "on-chip" bound proteins by microsequencing of the trypsin digested peptide fragments through tandem MS. Five mouse heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2/B1, A3, A/B, and D0 were identified. To verify those bound hnRNPs, a novel thioaptamer/antibody sandwich assay provides highly sensitive and selective identification of proteins on ProteinChip arrays.     

Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array.

Oligonucleotide microarrays, also called "DNA chips," are currently made by a light-directed chemistry that requires a large number of photolithographic masks for each chip. Here we describe a maskless array synthesizer (MAS) that replaces the chrome masks with virtual masks generated on a computer, which are relayed to a digital micromirror array. A 1:1 reflective imaging system forms an ultraviolet image of the virtual mask on the active surface of the glass substrate, which is mounted in a flow cell reaction chamber connected to a DNA synthesizer. Programmed chemical coupling cycles follow light exposure, and these steps are repeated with different virtual masks to grow desired oligonucleotides in a selected pattern. This instrument has been used to synthesize oligonucleotide microarrays containing more than 76,000 features measuring 16 microm 2. The oligonucleotides were synthesized at high repetitive yield and, after hybridization, could readily discriminate single-base pair mismatches. The MAS is adaptable to the fabrication of DNA chips containing probes for thousands of genes, as well as any other solid-phase combinatorial chemistry to be performed in high-density microarrays.     

Nuclear reorganization of DNA mismatch repair proteins in response to DNA damage

The DNA mismatch repair (MMR) system is highly conserved and vital for preserving genomic integrity. Current mechanistic models for MMR are mainly derived from in vitro assays including reconstitution of strand-specific MMR and DNA binding assays using short oligonucleotides. However, fundamental questions regarding the mechanism and regulation in the context of cellular DNA replication remain. Using synchronized populations of HeLa cells we demonstrated that hMSH2, hMLH1 and PCNA localize to the chromatin during S-phase, and accumulate to a greater extent in cells treated with a DNA alkylating agent. In addition, using small interfering RNA to deplete hMSH2, we demonstrated that hMLH1 localization to the chromatin is hMSH2-dependent. hMSH2/hMLH1/PCNA proteins, when associated with the chromatin, form a complex that is greatly enhanced by DNA damage. The DNA damage caused by high doses of alkylating agents leads to a G₂ arrest after only one round of replication. In these G₂-arrested cells, an hMSH2/hMLH1 complex persists on chromatin, however, PCNA is no longer in the complex. Cells treated with a lower dose of alkylating agent require two rounds of replication before cells arrest in G₂. In the first S-phase, the MMR proteins form a complex with PCNA, however, during the second S-phase PCNA is missing from that complex. The distinction between these complexes may suggest separate functions for the MMR proteins in damage repair and signaling. Additionally, using confocal immunofluorescence, we observed a population of hMSH6 that localized to the nucleolus. This population is significantly reduced after DNA damage suggesting that the protein is shuttled out of the nucleolus in response to damage. In contrast, hMLH1 is excluded from the nucleolus at all times. Thus, the nucleolus may act to segregate a population of hMSH2–hMSH6 from hMLH1–hPMS2 such that, in the absence of DNA damage, an inappropriate response is not invoked.     

Specific conjugation of DNA binding proteins to DNA templates through thiol-disulfide exchange

The double-stranded oligodeoxyribonucleotides with single internucleotide disulfide linkages were successfully used for covalent trapping of cysteine containing protein. In particular, an efficient conjugation of DNA methyltransferase SsoII to sequence-specific decoys was demonstrated. The obtained results assume that synthetic oligodeoxyribonucleotides bearing a new trapping site can be used as new tools to study and manipulate biological systems.     

Squaring up to DNA: pentapeptide repeat proteins and DNA mimicry

Pentapeptide repeats are a class of proteins characterized by the presence of multiple repeating sequences five amino acids in length. The sequences fold into a right-handed β-helix with a roughly square-shaped cross section. Pentapeptide repeat proteins include a number of examples which are thought to function as structural mimics of DNA and act to competitively bind to the type II topoisomerase DNA gyrase, an important antibacterial target. DNA gyrase-targeting pentapeptide repeat proteins can both inhibit DNA gyrase—a potentially useful therapeutic property—and contribute to resistance to quinolone antibacterials (by acting to prevent them forming a lethal complex with the DNA and enzyme). Pentapeptide repeat proteins are therefore of wide interest not only because of their unusual structure, function, and potential as an antibacterial target, but also because knowledge of their mechanism of action may lead to both a greater understanding of the details of DNA gyrase function as well as being a useful template for the design of new DNA gyrase inhibitors. However, many puzzling aspects as to how these DNA mimics function and indeed even their ability to act as DNA mimics itself remains open to question. This review summarizes the current state of knowledge regarding pentapeptide repeat proteins, focusing on those that are thought to mimic DNA, and speculates on potential structure-function relationships which may account for their differing specificities.     

Molecular-beacon-based array for sensitive DNA analysis

Molecular beacon (MB) DNA probes provide a new way for sensitive label-free DNA/protein detection in homogeneous solution and biosensor development. However, a relatively low fluorescence enhancement after the hybridization of the surface-immobilized MB hinders its effective biotechnological applications. We have designed new molecular beacon probes to enable a larger separation between the surface and the surface-bound MBs. Using these MB probes, we have developed a DNA array on avidin-coated cover slips and have improved analytical sensitivity. A home-built wide-field optical setup was used for imaging the array. Our results show that linker length, pH, and ionic strength have obvious effects on the performance of the surface-bound MBs. The fluorescence enhancement of the new MBs after hybridization has been increased from 2 to 5.5. The MB-based DNA array could be used for DNA detection with high sensitivity, enabling simultaneous multiple-target bioanalysis in a variety of biotechnological applications.     

Algorithmic approaches to selecting control clones in DNA array hybridization experiments

We study the problem of selecting control clones in DNA array hybridization experiments. The problem arises in the OFRG method for analyzing microbial communities. The OFRG method performs classification of rRNA gene clones using binary fingerprints created from a series of hybridization experiments, where each experiment consists of hybridizing a collection of arrayed clones with a single oligonucleotide probe. This experiment produces analog signals, one for each clone, which then need to be classified, that is, converted into binary values 1 and 0 that represent hybridization and non-hybridization events. In addition to the sample rRNA gene clones, the array contains a number of control clones needed to calibrate the classification procedure of the hybridization signals. These control clones must be selected with care to optimize the classification process. We formulate this as a combinatorial optimization problem called Balanced Covering. We prove that the problem is NP-hard, and we show some results on hardness of approximation. We propose approximation algorithms based on randomized rounding, and we show that, with high probability, our algorithms approximate well the optimum solution. The experimental results confirm that the algorithms find high quality control clones. The algorithms have been implemented and are publicly available as part of the software package called CloneTools.     

The Application of stereolithography to the fabrication of accurate molecular models

The process of stereolithography, which automatically fabricates plastic models from designs created in certain computer-aided design programs, has been applied to the production of accurate plastic molecular models. Atomic coordinates obtained from quantum mechanical calculations and from neutron diffraction data were used to locate spheres in the I-DEAS CAD program with radii proportional to the appropriate van der Waals radii. The sterolithography apparatus was used to build the models using a photosensitive liquid resin, resulting in hard plastic models that accurately represent the computed or experimental input structures. Three examples are given to illustrate how the models can be used to interpret experimental structure-activity data for systems of biological importance or host-guest chemistry: (1) Interpretation of kinetic data for the formation of a stable blocking complex between amiloride analogs and the epithelial sodium channel, (2) interpretation of binding and neural activity data for the interaction of certain amino acids and their analogs at the l-alanine taste receptor of the channel catfish, and (3) interpretation of shape selectivity and rate acceleration in cyclodextrin catalysis using models of the neutron diffraction structure of β-cyclodextrin and of the transition state for the cleavage of phenyl acetate by the secondary hydroxyl oxygen of β-cyclodextrin.