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1.
[graph: see text] We describe the synthesis of new environmentally sensitive fluorescence probes to elucidate DNA structures. DNA oligonucleotides containing fluorophore dan (6-(dimethylamino)-2-acylnaphthalene)-modified dC or dG were able to monitor the microenvironmental changes in both the major and minor grooves of DNA with a B- to A-DNA conformational transition and RNA hybridization. 相似文献
2.
Muris Kobašlija Andrew R. Bogdan Sarah L. Poe Fernando Escobedo D. Tyler Mcquade 《Journal of polymer science. Part A, Polymer chemistry》2008,46(7):2309-2315
Miscible solvents are phase separated within microcapsules containing entrapped polymers, thereby creating stable, isolated microenvironments. In particular, we demonstrate that encapsulated polyethyleneimine induces phase separation of toluene and methanol and that this phase separation is well modeled by the Flory‐Huggins theory. We further show the generality of microenvironment formation by phase separating tetrahydrofuran and acetone with entrapped polystyrene. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2309–2315, 2008 相似文献
3.
Ajo-Franklin CM Yoshina-Ishii C Boxer SG 《Langmuir : the ACS journal of surfaces and colloids》2005,21(11):4976-4983
Fluorescence interference contrast microscopy (FLIC) is a powerful method to structurally characterize fluorescent objects with nanometer-scale resolution in the z direction. Here we use FLIC to characterize the water layer underlying supported membranes and membrane-tethered double-stranded oligonucleotides. FLIC measurements of supported membranes containing the lipid-anchored fluorescent dye DiI in both leaflets indicate the thickness of the water layer separating the solid support and the lower lipid leaflet is 1.3 +/- 0.2 nm. Addition of cobalt(II) chloride to a DiI-supported membrane quenches the fluorescence in the top leaflet of the supported membrane; FLIC measurements of this system precisely locate the DiI to the bottom leaflet. These experiments confirm the accuracy of the model and parameters used to determine the water layer thickness, demonstrate the ability to differentiate between fluorescent objects whose average position differs by approximately 1.9 nm, and provide a widely applicable method to test the resolution of other high-z-resolution fluorescent microscopies. FLIC measurements of Alexa-labeled double-stranded oligonucleotides tethered to a supported membrane indicate that the DNA double helix is oriented perpendicular to the surface. Complications that arise from uncertainly in the orientation of the fluorophore are discussed. Several improvements in FLIC methodology are described. These stringent tests of the resolution of FLIC and the ability to unambiguously determine fluorescent lipid distribution provide structural insight on assemblies at membrane interfaces and permit the detection of even subtle changes at such interfaces. 相似文献
4.
Andersson M Elihn K Fromell K Caldwell KD 《Colloids and surfaces. B, Biointerfaces》2004,34(3):165-171
Colloidal polymer particles are widely used in a variety of applications ranging from chromatography to surface modified bioreactors in protein arrays. In the present study, surface attachment of polystyrene particles to a polystyrene substrate has been performed using oligonucleotide hybridization. Thiolated complementary oligomers of cytosine and guanine have been covalently coupled to a pyridyl disulphide (PDS) modified polyethyleneglycol tether, forming part of a triblock copolymer which is adsorbed to the polystyrene surfaces via hydrophobic polypropylene oxide center blocks. The ability to withstand shear forces was studied using a laminar flow cell and the uptake of oligomers on the particles was quantified using two complementary techniques: UV-spectroscopy and sedimentation field flow fractionation. The possibility to tether particles in a flow cell suitable for practical use in e.g. a FIA-system is demonstrated. 相似文献
5.
Jan Hoffmann Thorsten. L. Schmidt Alexander Heckel Bernd Brutschy 《Rapid communications in mass spectrometry : RCM》2009,23(14):2176-2180
In the present work we demonstrate the advantages of LILBID mass spectrometry (laser‐induced liquid bead ion desorption) in the analysis of nucleic acids and large oligonucleotides. For established methods like matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), the mass analysis of oligonucleotides or of noncovalent oligonucleotide‐protein complexes, in particular of very large ones, still represents a considerable challenge either due to the lack of native solutions or nonspecific adduct formation or due to a reduced salt tolerance or a high charge state of the ions. With LILBID, oligonucleotides, solvated in micro‐droplets of aqueous buffer at certain pH and ion strength, are brought into the gas phase by laser ablation. We show that our method is able to detect single‐ and double‐stranded oligonucleotides with high softness, demonstrated by the buffer dependence of the melting of a duplex. The absolute sensitivity is in the attomole range concomitant with a total analyte consumption in the femtomole region. The upper mass limit of oligonucleotides still detected with good signal‐to‐noise ratio with LILBID is the 1.66 MDa plasmid pUC19. With DNA ladders from short duplexes with sticky ends, we show that LILBID correctly reflects the relative thermodynamic stabilities of the ladders. Moreover, as an example for a specific DNA–protein complex we show that a NF‐κB p50 homodimer binds sequence specifically to its match DNA. In summary we demonstrate that LILBID, although presently performed only with low mass resolution, due to these advantages, is an alternative mass spectrometric method for the analysis of oligonucleotides in general and of specific noncovalent nucleic acid–protein complexes in particular. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
6.
Jennifer A. Dougan 《Tetrahedron letters》2010,51(44):5787-5790
The H-phosphonate of a derivative of thioctic acid (TA) was synthesised and used to introduce a disulfide moiety at the 5′-end of oligonucleotides. This method overcomes the difficulties experienced with the phosphoramidite approach when employing a cyclic disulfide in the starting alcohol. The disulfide-modified oligonucleotides are subsequently used in metallic nanoparticle (Au and Ag) and surface functionalisation for sensitive, sequence specific analytical detection strategies. 相似文献
7.
Summary Capillary gel electrophoresis (CGE) has been recognized as an effective method for the analysis of oligonucleotides. CGE using
polymer solutions is especially useful and effective compared with that using crosslinked gels, because of easy change of
media. Replacement of media leads to the reproducible separation of analytes. We have investigated CGE analysis of oligonucleotides
of less than 20 bases employing various kinds of polymers. Polyacrylamide, dextrin, dextran, pullakin, and poly(ethylene glycol)
were used as sieving matrixes at concentrations of 0–30 %. Polydeoxythymidylic acids [p(dT)11–20] were used as a test sample. These small oligonucleotides were successfully resolved on the basis of their base number by
CGE using some of these polymer solutions. In particular, dextran was found to be effective and baseline separation was observed
when a 30 % dextran solution was employed. Some validations such as linearity and reproducibility were also established and
this method was found to be an adequate quality control method for small oligonucleotides. Finally, CGE using a 30 % dextran
solution was successfully applied to impurity profiling of some synthetic oligonucleotides. 相似文献
8.
Oligonucleotides have developed into highly versatile and selective therapeutics over the past 20 years. More than five discrete mechanisms of action have been reported and more than 10 different chemical modifications have been used to extend their in vivo half-life and reduce their toxicity. Capillary gel electrophoresis (CGE) has been used extensively for the quantitative analysis of oligonucleotide therapeutics in both preclinical and clinical studies since the 1990s. The success of CGE is based on its extraordinary resolving power, which allows for the simultaneous determination of the parent drug and its metabolites. More recently, capillary gel electrophoresis has seen renewed interest with the emergence of replaceable gels with single-base resolving power and new capillary electrophoresis-mass spectrometry interfaces. This review discusses the bioanalysis of therapeutic oligonucleotides showing the evolution of the field over the past two decades leading to the current new approaches. Included in this review are topics such as different gel types, sample introduction modes, sample extraction procedures, separation conditions and detection methods used in CGE, along with discussions of the successes and limitations associated with each. 相似文献
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10.
Hagan NA Smith CA Antoine MD Lin JS Feldman AB Demirev PA 《Journal of the American Society for Mass Spectrometry》2012,23(4):773-777
The capability to rapidly and confidently determine or confirm the sequences of short oligonucleotides, including native and
chemically-modified DNA and RNA, is important for a number of fields. While matrix-assisted laser desorption/ionization (MALDI)
time-of-flight (TOF) mass spectrometry (MS) has been used previously to sequence short oligonucleotides, the typically low
fragmentation efficiency of in-source or post-source decay processes necessitates the accumulation of a large number of spectra,
thus limiting the throughput of these methods. Here we introduce a novel matrix, 1,5-diaminonapthalene (DAN), for facile in-source
decay (ISD) of DNA and RNA molecular anions, which allows for rapid sequence confirmation. d-, w-, and y-series ions are prominent in the spectra, complementary to the (a-B)- and w- ions that are typically produced by MALDI post-source decay (PSD). Results are shown for several model DNA and RNA oligonucleotides,
including combinations of DAN-induced fragmentation with true tandem TOF MS (MS/MS) for pseudo-MS3 and “activated-ion PSD.” 相似文献
11.
A variety of techniques exist that provide chemical information in the form of a spatially resolved image. For example, magnetic resonance imaging has significant advantages for biological applications because it allows measurements in intact tissues. However, it is restricted at the present time to species of high concentration. The most common approach for chemically selective imaging is to use optical microscopy coupled with fluorescence measurements. A variety of fluorophores with high selectivity for specific ions are commercially available and are readily used in such applications. While imaging techniques provide an overall view of the microenvironment, probes that give a high fidelity view of a specific species and its concentration at a single location are also useful. Thus, there is particular interest in developing local sampling techniques. Electrochemical techniques, both potentiometric and amperometric, are particularly useful in this regard. For example, neurotransmitters, compounds secreted from nerve terminals to relay information from one neuron to another, can readily be examined with electrodes of micrometer dimensions. In this way, the mechanisms that control the secretion and the regulation of these compounds in the extracellular space can be evaluated. These tools can also be used at the level of single cells, and when placed at different locations, can be used to provide a chemical image. This approach essentially represents a marriage of electrochemical techniques with those of the scanning microscopies, an area that will see considerable growth in the new millennium. 相似文献
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13.
To investigate the photoreactions of BrU in Z-DNA, the photoirradiation of 5'-d(C1G2C3G4BrU5G6C7G8)-3'/5'-d(C9mG10C11A12C13mG14C15G16)-3'(ODN 1-2) was investigated. In accord with previous observations, B-form ODN 1-2 with the 5'-GBrU sequence showed very weak photoreactivity. However, Z-form ODN 1-2 in 2 M NaCl underwent photoreaction to afford 5'-d(CGC)rGd(UGCG)-3' together with the formation of imidazolone (Iz) contained 5'-d(CIzCACmGCG)-3'. The results clearly indicate that structural changes caused by the B-Z transition dramatically increased the photoreactivity of ODN 1-2. Inspection of the molecular structure of Z-DNA suggests that there is unique four-base pi-stacks at the G4-BrU5-C11-mG10 in ODN 1-2. These results suggest that the intriguing possibility that the mG10 in a complementary strand located at the end of the four-base pi-stacks may act as an electron donor. To test the hypothesis of interstrand charge transfer from mG10 to BrU5 within the four-base pi-stacks in Z-DNA, ODN 1-3 samples in which the putative donor G10 residue was replaced with 8-methoxyguanine (moG) were prepared, since moG is known to trap cation radicals to yield Iz moieties in DNA. Photoirradiation of ODN 1-3 efficiently produced 5'-d(CGC)rGd(UGCG)-3' together with formation of 5'-d(CIzCACmGCG)-3'. These results clearly indicate that the interstrand charge transfer from mG10 to BrU5 initiates the photoreaction. In clear contrast, other replacements of G with moG did not enhance the photoreactivity. The present study revealed the presence of unique four-base pi-stacks in Z-DNA and photoirradition of BrU in Z-DNA causes efficient electron transfer from G within this cluster. 相似文献
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16.
A new system for the colorimetric detection of oligonucleotides was developed using polydiacetylene vesicles, which play the dual role of an indicator of color transition and an amplification tag. The results are of significance in understanding the mechanism of color transition of biological recognition in polydiacetylene systems and in designing new biosensors. 相似文献
17.
We describe a coarse-grained model for Z-DNA that mimics the DNA shape with a relatively small number of repulsive interaction sites. In addition, negative charges are placed at the phosphate positions. The ionic atmosphere around this grooved Z-DNA model is then investigated with Monte Carlo simulation. Cylindrically averaged concentration profiles as well as the spatial distribution of ions have been calculated. The results are compared to those for other DNA models differing in the repulsive core. This allows the examination of the effect of the DNA shape in the ionic distribution. It is seen that the penetrability of the ions to the DNA groove plays an important role in the ionic distribution. The results are also compared with those reported for B-DNA. In both conformers the ions are structured in alternating layers of positive and negative charge. In Z-DNA the layers are more or less concentric to the molecular axis. Besides, no coions enter into the single groove of this conformer. On the contrary, the alternating layers of B-DNA are also structured along the axial coordinate with some coions penetrating into the major groove. In both cases we have found five preferred locations of the counterions and two for the coions. The concentration of counterions reaches its absolute maximum at the narrow Z-DNA groove and at the minor groove of B-DNA, the value of the maximum being higher in the Z conformer. 相似文献
18.
Electrochemical determination of melamine using oligonucleotides modified gold electrodes 总被引:1,自引:0,他引:1
A novel and simple electrochemical method for determination of melamine is developed based on oligonucleotides film modified gold electrodes. The electrochemical probe of ferricyanide was used to investigate the interactions between oligonucleotides and melamine. Results of cyclic voltammetries, differential pulse stripping voltammetries, electrochemical impedance spectrometry and atomic force microscope, proved that melamine might interact with oligonucleotides mainly through electrostatic and hydrogen-bonding interactions. The interactions between oligonucleotides and melamine lead to the increase in the peak currents of ferricyanide, which could be used for electrochemical sensing of melamine. The redox peak currents of ferricyanide were linear with the concentration of melamine in the range from 3.9 × 10−8 to 3.3 × 10−6 M with a linear coefficiency of 0.990. The detection limit was 9.6 × 10−9 M. The proposed electrochemical biosensor is rapid, convenient and low-cost for effective sensing of melamine. Particularly, the proposed method was applied successfully to the determination of melamine in milk products, and the recovery was 95%. 相似文献
19.
Understanding the interaction between soluble factors and cells in the cellular microenvironment is critical to understanding a wide range of diseases. Microchannel culture systems provide a tool for separating diffusion and convection based transport making possible controlled studies of the effects of soluble factors in the cellular microenvironment. In this paper we compare the proliferation kinetics of cells in traditional culture flasks to those in microfluidic channels, and explore the relationship between microchannel geometry and cell proliferation. PDMS (polydimethylsiloxane) microfluidic channels were fabricated using micromolding methods. Fall armyworm ovarian cells (Sf9) were homogeneously seeded in a series of different sized microchannels and cultured under a no flow condition. The proliferation rates of Sf9 cells in all of the microchannels were slower than in the flask culture over the first 24 h of culture. The proliferation rates in the microchannels then continuously decreased reaching 5% of that in the flasks over the next 48 h and maintained this level for 5 days. This growth inhibition was reversible and influenced only by the cell seeding density and the channel height but not the channel length or width. One possible explanation for the observed dimension-dependent cell proliferation is the accumulation of different functional molecules in the diffusion dominant microchannel environment. This study provides insights into the potential effects of the diffusion of soluble factors and related effects on cell behavior in microenvironments relevant to the emerging use of microchannel culture systems. 相似文献
20.
Residual dipolar couplings were successfully used to distinguish between the two diastereotopic protons on C-20 of strychnine dissolved in an organic liquid crystal (PBLG/CDCl(3)). The results presented here strongly suggest that this method will be of help in organic structure determination, making the determination of relative stereochemistry in the absence of NOE data possible. 相似文献