分子信标DNA计算模型的研究进展与展望

由于分子信标具有结构简单,灵敏度高及反应迅速等优点,因此,利用分子信标进行数学问题的求解将成为可能.通过对分子信标的计算模型进行详细的介绍,并对分子信标的计算模型的研究思路进行了展望,据此思路,可以建立多种组合优化问题及逻辑门的分子信标计算模型.     

A microassay for detection of DNA and RNA in small numbers of plant cells

Summary A microtechnique for the detection of DNA or RNA in small numbers of plant cells (1–50) has been developed using cauliflower mosaic virus (CaMV) infection of turnip as a model system. Both DNA and RNA extracted from 10 mesophyll protoplasts from CaMV-infected plants can be detected by hybridization using a radioactive probe made from cloned CaMV DNA (pCaMV10). No hybridization above background was detected in extracts of protoplasts from uninfected plants. At least 0.15 pg (11 000 molecules) of purified pCaMV10 DNA can be detected. This method is superior to existing macro techniques for nucleic acid detection as smaller amounts of tissue are required and the detection is approximately 100-fold more sensitive. re]19850326 rv]19850530 ac]19850611     

Fano Resonance Excited All-Optical XOR,XNOR, and NOT Gates with High Contrast Ratio

We have presented all-optical XOR, XNOR, and NOT gates using metal-insulator-metal (MIM)-coupled ring resonator. The performance of the device is evaluated by finite difference in time-domain (FDTD) method. The proposed gate utilizes a unique phenomenon of Fano resonance to excite logic OFF/ON state. Fano resonance has quite asymmetric resonance profile and the transmission spectrum of Fano profile abruptly drops to a minimum value at the resonance condition. Due to this unique resonance phenomenon, a large value of contrast ratio is obtained. The proposed XNOR gate offers a contrast ratio (C.R.) of 20.66 dB while XOR and NOT gates offer C.R. 12.8 and 18.8 dB respectively. The variation of contrast ratio is also studied against different input wavelength and it is reported that the obtained value of contrast ratio is an optimum value for the proposed structure. The device is compact sized with small dimension 0.31 λ₀², where λ₀?=?1.55 μm. The proposed device opens up the avenues for designing on-chip optical gates in the field of high-speed optical communication networks.     

Chemical genomics and medicinal systems biology: chemical control of genomic networks in human systems biology for innovative medicine

With advances in determining the entire DNA sequence of the human genome, it is now critical to systematically identify the function of a number of genes in the human genome. These biological challenges, especially those in human diseases, should be addressed in human cells in which conventional (e.g. genetic) approaches have been extremely difficult to implement. To overcome this, several approaches have been initiated. This review will focus on the development of a novel "chemical genetic/genomic approach" that uses small molecules to "probe and identify" the function of genes in specific biological processes or pathways in human cells. Due to the close relationship of small molecules with drugs, these systematic and integrative studies will lead to the "medicinal systems biology approach" which is critical to "formulate and modulate" complex biological (disease) networks by small molecules (drugs) in human bio-systems.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

Probe amplifier system based on chimeric cycling oligonucleotides

Amplification systems are required as part of DNA probe technology, since traditional non-amplified oligonucleotide hybridization using nonradioactive detection methods have detection limits of approximately 10(8) molecules. We present a probe amplifier technology suitable for use in large-scale automated clinical diagnostic systems. It is fast, sensitive and performs at a constant temperature. The system functions by allowing a single target molecule to act as a catalyst in converting a large number of probe molecules to a unique detectable form. We refer to this catalytic amplification process as the "cycling probe reaction." The basis of the system is an oligomer probe construction consisting of a DNA-RNA-DNA sequence.     

Small molecule induction of MSH2-dependent cell death suggests a vital role of mismatch repair proteins in cell death

Avoidance of apoptosis is one of the hallmarks of cancer development and progression. Chemotherapeutic agents aim to initiate an apoptotic response, but often fail due to dysregulation. MSH proteins are capable of recognizing cisplatin damage in DNA and participate in the initiation of cell death. We have exploited this recognition and computationally simulated a MutS homolog (MSH) "death conformation". Screening and docking experiments based on this model determined that the MSH2-dependent cell-death pathway can be induced by a small molecule without DNA damage, reserpine. Reserpine was identified via virtual screening on structures obtained from molecular dynamics as a small molecule that selectively binds a protein "death" conformation. The virtual screening predicts that this small molecule binds in the absence of DNA. Cell biology confirmed that reserpine triggers the MSH2-dependent cell-death pathway. This result supports the hypothesis that the MSH2-dependent pathway is initiated by specific protein conformational changes triggered by binding to either DNA damage or small compound molecules. These findings have multiple implications for drug discovery and cell biology. Computational modeling may be used to identify and eventually design small molecules that selectively activate particular pathways through conformational control. Molecular dynamics simulations can be used to model the biologically relevant conformations and virtual screening can then be used to select for small molecules that bind specific conformations. The ability of a small molecule to induce the cell-death pathway suggests a broader role for MMR proteins in cellular events, such as cell-death pathways, than previously suspected.     

Detection of C677T mutation of MTHFR in subject with coronary heart disease by hairpin probe with enzymatic color on microarray

Molecular beacon (MB) is especially suited for detection of single nucleotide polymorphism (SNP), and the type of MB immobilized on the surface of microarray in particular, may detect multi-sample and multi-locus. However, the majority of MB needs to be labeled with fluorescence and quenching molecules on the two ends of the probe, and observed the reaction of fluorescence or complicated electrochemical signal produced hybridization of MB and target sequence by complex and expensive instruments. The "molecular beacon" and microarray designed appropriately in our study can produce visible light response signal induced by amplification effect of enzymatic color, and are avoided with the marker of fluorescence and quenching molecules and expensive instruments. The "molecular beacon" without fluorescence and quenching molecules is entitled as "hairpin DNA probe" by us for only the "hairpin" structure of traditional molecular beacon is adopted. The merits of two techniques, molecular beacon and amplification effect of enzymatic color, are successfully combined, and the technique is simple, sensitive and specific, to detect and compare the methylenetetrahydrofolate reductase (MTHFR) Gene C677T mutation of subjects between coronary heart disease (CHD) and control group. The results showed that MTHFR Gene C677T polymorphism is an independent risk factor for CHD.     

An FET-type charge sensor for highly sensitive detection of DNA sequence

We have fabricated an field effect transistor (FET)-type DNA charge sensor based on 0.5 microm standard complementary metal oxide semiconductor (CMOS) technology which can detect the deoxyribonucleic acid (DNA) probe's immobilization and information on hybridization by sensing the variation of drain current due to DNA charge and investigated its electrical characteristics. FET-type charge sensor for detecting DNA sequence is a semiconductor sensor measuring the change of electric charge caused by DNA probe's immobilization on the gate metal, based on the field effect mechanism of MOSFET. It was fabricated in p-channel (P) MOSFET-type because the phosphate groups present in DNA have a negative charge and this charge determines the effective gate potential of PMOSFET. Gold (Au) which has a chemical affinity with thiol was used as the gate metal in order to immobilize DNA. The gate potential is determined by the electric charge which DNA possesses. Variation of the drain current versus time was measured. The drain current increased when thiol DNA and target DNA were injected into the solution, because of the field effect due to the electrical charge of DNA molecules. The experimental validity was verified by the results of mass changes detected using quartz crystal microbalance (QCM) under the same measurement condition. Therefore it is confirmed that DNA sequence can be detected by measuring the variation of the drain current due to the variation of DNA charge and the proposed FET-type DNA charge sensor might be useful in the development for DNA chips.     

Detection of hybrid formation between peptide nucleic acids and DNA by fluorescence polarization in the presence of polylysine.

A new method for the detection of PNA/DNA hybrids is presented. In this method, short PNA probes (9-13 mer) are labeled with a fluorescent dye and allowed to hybridize to target DNA molecules. A cationic polyamino acid, such as polylysine, is then added to the reaction mixture, whereupon the DNA molecules bind electrostatically to this polycation. The PNA probes, which are uncharged or may carry only a small charge due to the fluorescent dye, do not bind to polylysine unless hybridized to the negatively charged DNA target. The binding of the labeled PNA/DNA hybrid to the high-molecular-weight polymer leads to a significant change in the rotational correlation time of the fluorophore attached to the PNA. This can be conveniently detected by measuring the fluorescence polarization of the latter. The method is completely homogeneous because no separation of free from bound PNA probe is required. The hybridization and dehybridization reactions can be followed in real time. The method has been applied to the typing of single-nucleotide polymorphisms in PCR products.     

Digital mapping of bacterial artificial chromosomes by fluorescence in situ hybridization

The bacterial artificial chromosome (BAC) has become the most popular tool for cloning large DNA fragments. The inserts of most BAC clones average 100-200 kilobases (kb) and molecular characterization of such large DNA fragments is a major challenge. Here we report a simple and expedient technique for physical mapping of BAC inserts. Individual BAC molecules were immobilized on glass slides coated with Poly-L-lysine. The intact circular BAC molecules were visualized by fluorescence in situ hybridization using BAC DNA as a probe. The 7.4 kb BAC vector was extended to approximately 2.44 kb per micrometer. Digitally measured linear distances can be transformed into kilobases of DNA using the extension of BAC vector as a standard calibration. We mapped DNA fragments as small as 2 kb directly on circular BAC molecules. A rice BAC clone containing both tandem and dispersed repeats was analyzed using this technique. The distribution and organization of the different repeats within the BAC insert were efficiently determined. The results showed that this technique will be especially valuable for characterizing BAC clones that contain complex repetitive DNA sequences.     

Physicochemical perspectives on DNA microarray and biosensor technologies

Detection and sequence-identification of nucleic acid molecules is often performed by binding, or hybridization, of specimen "target" strands to immobilized, complementary "probe" strands. A familiar example is provided by DNA microarrays used to carry out thousands of solid-phase hybridization reactions simultaneously to determine gene expression patterns or to identify genotypes. The underlying molecular process, namely sequence-specific recognition between complementary probe and target molecules, is fairly well understood in bulk solution. However, this knowledge proves insufficient to adequately understand solid-phase hybridization. For example, equilibrium binding constants for solid-phase hybridization can differ by many orders of magnitude relative to solution values. Kinetics of probe-target binding are affected. Surface interactions, electrostatics and polymer phenomena manifest themselves in ways not experienced by hybridizing strands in bulk solution. The emerging fundamental understanding provides important insights into application of DNA microarray and biosensor technologies.     

A model for intramolecular recombination of herpesvirus DNA leading to the formation of circular, circular-linear and fragmented genomes.

A model is presented for intramolecular recombination of herpesvirus DNA. It is proposed that the terminal repeat sequences of the viral DNA contain insertion sequences which may integrate with homologous repeat sequences between the long (L) and short (S) components. In class 2 herpes-virus DNA (as defined by Honess &; Watson, 1977) in which the repeat sequences flank the S component only, circular-linear DNA molecules can be formed as an intermediate step. Reorientation of the S component leads to the formation of two DNA isomers. In class 3 herpesvirus DNA in which repeat sequences flank both the L and S components, either circular-linear or 8-shaped DNA molecules are proposed as intermediates leading to the formation of four DNA isomers. Fragmentation of the S component could lead to the formation of small circular DNA molecules.     

Restriction endonuclease SsoII with photoregulated activity--a "molecular gate" approach

A novel method for regulating the activity of homodimeric proteins--"molecular gate" approach--was proposed and its usefulness illustrated for the type II restriction endonuclease SsoII (R.SsoII) as a model. The "molecular gate" approach is based on the modification of R.SsoII with azobenzene derivatives, which allows regulating DNA binding and cleavage via illumination with light. R.SsoII variants with single cysteine residues introduced at selected positions were obtained and modified with maleimidoazobenzene derivatives. A twofold change in the enzymatic activity after illumination with light of wavelengths of 365 and 470 nm, respectively, was demonstrated when one or two molecules of azobenzene derivatives were attached to the R.SsoII at the entrance of or within the DNA-binding site.     

Amplification of PFGE-separated chromosome-specific DNA by degenerate oligonucleotide-primed PCR (DOP-PCR)

Specific probes for DNA molecules of high molecular weight can be efficiently and rapidly prepared by the combination of pulsed-field gel electrophoresis (PFGE) with degenerate oligonucleotide-primed PCR (DOP-PCR). More than 10 mg of a specific probe were amplified from only 1 ng of a PFGE-separated DNA fragment of a red alga, Cyanidioschyzon merolae. The probe hybridized specifically to the fragment which was used as a template for DOP-PCR.     

Ancient DNA Damage

Under favorable conditions DNA can survive for thousands of years in the remains of dead organisms. The DNA extracted from such remains is invariably degraded to a small average size by processes that at least partly involve depurination. It also contains large amounts of deaminated cytosine residues that are accumulated toward the ends of the molecules, as well as several other lesions that are less well characterized.In living cells, DNA molecules continuously suffer chemical insults, which are countered by enzymatic repair mechanisms that maintain the integrity of the genome (Lindahl 1993). On death, these cellular repair mechanisms cease to function. As a consequence, the genome becomes exposed to the unmitigated effects of numerous factors that threaten its stability. These factors include intracellular nucleases, which are no longer sequestered in the cell and can thus gain access to DNA and degrade it, as well as microorganisms that spread in the decaying tissues. Together these factors may lead to the loss of all retrievable DNA. However, under favorable environmental conditions, for example when tissues are frozen or become desiccated quickly after death, these processes become inhibited before the complete destruction of all DNA endogenous to the organism. In these instances other destructive factors, particularly hydrolytic and oxidative processes, become limiting to the time that DNA survives in a tissue.When DNA is extracted and analyzed from ancient samples these destructive factors manifest themselves in three different ways: (i) a reduction in DNA fragment size, (ii) lesions that block the replication of the DNA molecules by polymerases, thus impeding many forms of analysis, and (iii) lesions that cause incorrect nucleotides to be incorporated when the DNA is replicated. Here, we summarize what is known about each of these forms of damage in ancient DNA.     

DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures

Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 m. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4–6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4–6S) and large (20–30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm³. Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 m.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.Abbreviations BUdR 5-Bromodeoxyuridine - FUdR 5-Fluorodeoxyuridine