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1.
Wochner A Menger M Orgel D Cech B Rimmele M Erdmann VA Glökler J 《Analytical biochemistry》2008,373(1):34-42
We describe the characterization of a DNA aptamer that displays high affinity and specificity for the anthracyclines daunomycin and doxorubicin, both of which are frequently used in chemotherapy. Aptamers were isolated from a pool of random sequences using a semiautomated procedure for magnetic beads. All selected aptamers displayed high affinity for the target molecule daunomycin. One aptamer was further characterized and exhibited a dissociation constant (KD) of 20 nM. To examine the aptamer's binding properties and clarify its applicability for diagnostic assays, its performance under various buffer conditions was evaluated. The aptamer proved to be very robust and not dependent on the presence of specific ions. It also tolerated a wide pH range and immobilization via 5'-biotinylation. Furthermore, a competition assay for sensitive daunomycin detection was established. This not only allows the determination of the aptamer's specificity but also allows the quantification of as little as 8.4 microg/L daunomycin and doxorubicin. 相似文献
2.
The generation of molecules that selectively recognize specific conformations of a protein is an important component of the elucidation protein function. We have used SELEX (Systematic Evolution of Ligands by EXponential enrichment) technology to produce aptamers that bind in a conformationally selective manner to calsenilin, which involved in Ca2+-mediated apoptotic signaling. Since the conformations of calsenilin are quite different in the presence and absence of Ca2+, aptamers were selected against the dimeric protein both under calcium-bound and calcium-free conditions. We have found that aptamer-12 selectively binds to the dimeric form of the protein in the presence of calcium ion, while the binding of aptamer-2 does not discriminate between the Ca2+ bound and unbound protein. Data obtained from biochemical and biophysical experiments suggest that a dominant conformation of calcium-bound calsenilin exists in one dominant conformation and that one aptamer can be generated to recognize this conformation. In addition, observation made in this effort that aptamers selected against the two different conformations of calsenilin have different characteristics suggest that aptamers can serve as a plausible tool for recognizing various conformations of proteins, even those caused by interactions with small molecules or ions such as Ca2+. 相似文献
3.
Bioinformatic Analysis of the Contribution of Primer Sequences to Aptamer Structures 总被引:1,自引:0,他引:1
Aptamers are nucleic acid molecules selected in vitro to bind a particular ligand. While numerous experimental studies have examined the sequences, structures, and functions of individual aptamers, considerably fewer studies have applied bioinformatics approaches to try to infer more general principles from these individual studies. We have used a large Aptamer Database to parse the contributions of both random and constant regions to the secondary structures of more than 2000 aptamers. We find that the constant, primer-binding regions do not, in general, contribute significantly to aptamer structures. These results suggest that (a) binding function is not contributed to nor constrained by constant regions; (b) in consequence, the landscape of functional binding sequences is sparse but robust, favoring scenarios for short, functional nucleic acid sequences near origins; and (c) many pool designs for the selection of aptamers are likely to prove robust. 相似文献
4.
Aptamers are functional nucleic acids possessing high affinity and specificity to their cognate ligands and are isolated from a library of nucleic acids by iterative rounds of selection and amplification. In the current study, we used surface plasmon resonance (Biacore) as an efficient methodology for selecting aptamers that bind to hemagglutinin (HA) of human influenza virus. This procedure allowed us to monitor and select the target-bound aptamers specifically and simultaneously. These studies not only yielded an aptamer that binds to the HA of influenza virus with high affinity but also revealed the consensus sequence, 5'-GUCGNCNU(N)(2-3)GUA-3, for HA recognition. 相似文献
5.
A synthetic library of ca. 1013 single stranded oligodeoxynucleotides, each comprising a randomized 40mer sequence and homogeneous 10mer flanking regions, was screened for binding to recombinant human 14-3-3γ. A single aptamer, which showed similar affinities (KD ∼ 10−8 M) for six isoforms of the protein, has been shown to bind to undenatured 14-3-3 protein in the cerebral spinal fluid of scrapie infected sheep. 相似文献
6.
Björn Niebel Christine I. Wosnitza Michael Famulok 《Bioorganic & medicinal chemistry》2013,21(20):6239-6246
Rho GTPases regulate the actin cytoskeleton and thereby control cell migration, cell morphology, cell motility, and other cellular functions. The gene product of the oncogene Tiam1 acts as a guanine nucleotide exchange factor (GEF) for the Rho GTPase Rac. Like other RhoGEFs, Tiam1 is involved in cancer progression, but it also counteracts invasion in different cancer cell types. Hence, further investigations are required to unravel the functions of Tiam1 in the context of cancer initiation and progression, which appear to be cell specific. Although RhoGEFs in general seem to be attractive therapeutic targets, not many inhibitors have been described, yet. Here we report the identification and characterization of inhibitory RNA aptamers that specifically target Tiam1. After 16 selection rounds three aptamers sharing a 15 nucleotides consensus motif were identified. The clones K91 and K11 inhibited the Tiam1-mediated activation of the GTPase Rac2 in vitro. The tightest binder K91 neither bound the Rho GEF Vav1 nor the Arf GEF Cytohesin-2. In the presence of Rac1, the binding of K91 to Tiam1 was impaired indicating that the binding motif on Tiam1 overlaps with the GTPase binding site. K91 and K11 are the first reported inhibitory molecules targeting the GEF function of Tiam1. Due to their specificity over related GEF proteins they may represent promising tools for further elucidation of the biological functions of Tiam1. We anticipated that these aptamers will prove useful in validating the ambiguous roles of Tiam1 in cancer biology. 相似文献
7.
Jeffrey E. Vandenengel 《Biochemical and biophysical research communications》2009,378(1):51-733
Structure-switching signaling aptamers are nucleic acids that change shape upon binding to a specific ligand. Previously, we applied a new in vitro selection strategy to isolate structure-switching RNA aptamers responsive to the aminoglycoside antibiotic tobramycin. Here, we report the results of mutational analysis, secondary structure modeling, and ligand-specificity studies that suggest a mechanism for tobramycin-triggered structure switching. 相似文献
8.
9.
Our objective was to create a novel fluorogenic substrate for efficient in vitro kinetic assays on caspase-3. We designed a TAMRA (5′-tetramethylrhodamine-5(6)-carboxamide)- and Cy5 (cyanine 5)-labeled probe that allowed us to evaluate the caspase-3 activity via the changes in fluorescence intensity and wavelength. The prepared probe was found to be an efficient and selective substrate of caspase-3, with Vmax of 41.4 ± 3.3 nM/min and KM of 1.60 ± 0.23 μM. The strategy used in the design of this fluorogenic substrate can be applied in future endeavors to development of substrates for caspase-3 inhibitor screening assays or for real-time detection of apoptosis in living cells. 相似文献
10.
Swee Yang Low Jordan Peccia 《Biochemical and biophysical research communications》2009,386(3):544-328
Allergies are caused by the binding of IgE antibodies onto specific sites on allergens. However, in the assessment of exposure to airborne allergens, current techniques such as whole spore counts fail to account for the presence of these allergenic epitopes that trigger allergic reactions. The objective of the research is to develop a DNA aptamer for the Asp f 1 allergen of the pathogenic fungus Aspergillus fumigatus, using an IgE-binding epitope of the allergen as the target for aptamer selection. Through in vitro SELEX, an aptamer has been produced that binds with nanomolar affinity to the Asp f 1 IgE-epitope. The aptamer is also able to recognize the native Asp f 1 allergen, and does not bind to allergenic proteins from non-target mold species such as Alternaria alternata. Production of this aptamer provides proof-of-principle that allergen measurement methods can be developed to indicate the potent fraction, or allergenicity, of allergens. 相似文献
11.
Targeted inhibition of alphavbeta3 integrin with an RNA aptamer impairs endothelial cell growth and survival 总被引:1,自引:0,他引:1
Mi J Zhang X Giangrande PH McNamara JO Nimjee SM Sarraf-Yazdi S Sullenger BA Clary BM 《Biochemical and biophysical research communications》2005,338(2):956-963
Alphavbeta3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. Alphavbeta3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of alphavbeta3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-alphavbeta3 that binds recombinant alphavbeta3 integrin, for its ability to bind endogenous alphavbeta3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-alphavbeta3 binds alphavbeta3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-alphavbeta3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-alphavbeta3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-alphavbeta3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation. 相似文献
12.
Thermodynamic stability and mutational robustness of secondary structure are critical to the function and evolutionary longevity of RNA molecules. We hypothesize that natural and artificial selection for functional molecules favors the formation of structures that are stable to both thermal and mutational perturbation. There is little direct evidence, however, that functional RNA molecules have been selected for their stability. Here we use thermodynamic secondary structure prediction algorithms to compare the thermal and mutational robustness of over 1000 naturally and artificially evolved molecules. Although we find evidence for the evolution of both types of stability in both sets of molecules, the naturally evolved functional RNA molecules were significantly more stable than those selected in vitro, and artificially evolved catalysts (ribozymes) were more stable than artificially evolved binding species (aptamers). The thermostability of RNA molecules bred in the laboratory is probably not constrained by a lack of suitable variation in the sequence pool but, rather, by intrinsic biases in the selection process. 相似文献
13.
Xiaolei Pan Kavita A. Iyer Hebing Liu Douglas H. Sweet Małgorzata Dukat 《Bioorganic & medicinal chemistry letters》2017,27(18):4440-4445
Human organic cation transporters (OCTs) represent an understudied neurotransmitter uptake mechanism for which no selective agents have yet been identified. Several neurotransmitters (e.g. serotonin, norepinephrine) are low-affinity substrates for these transporters, but possess higher affinity for other transporters (e.g. the serotonin or norepinephrine transporters; SERT and NET, respectively). We have identified a new class of OCT inhibitors with a phenylguanidine structural scaffold. Here, we examine the actions of a series of such compounds and report preliminary structure–activity relationships (SARs) – the first dedicated SAR study of OCT3 action. Initial results showed that the presence of a substituent on the phenyl ring, as well as its position, contributes to the phenylguanidines’ inhibitory potency (IC50 values ranging from 2.2 to >450 μM) at hOCT3. There is a trend towards enhanced inhibitory potency of phenylguanidines with increased lipophilic character and the size of the substituent at the phenyl 4-position, with the latter reaching a ceiling effect. The first PiPT-based hOCT3 homology models were generated and are in agreement with our biological data. 相似文献
14.
Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells 总被引:6,自引:0,他引:6
Yu-Yau Joanne Shyr Angus G. Hepburn Jack M. Widhohn 《Molecular & general genetics : MGG》1992,232(3):377-382
Summary CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- DTT
dithiothreitol
- EPSPS
5-enolpyruvylshikimate-3-phosphate synthase
- I50
50% inhibitory concentration
- Kb
Kilobase (pairs)
- PEP
phosphoenolpyruvate
- PMSF
phenylmethylsulfonyl fluoride
- PVPP
polyvinylpolypyrrolidone
- S-3-P
shikimate-3-phosphate 相似文献
15.
The ribosome is the ribonucleoprotein particle responsible for translation of genetic information into proteins. The RNA component
of the ribosome has been implicated as the catalytic entity for peptide bond formation based on protease resistance and structural
data indicating an all-RNA active site. Nevertheless, peptidyl transfer by ribosomal RNA (rRNA) alone has not been demonstrated.
In an attempt to show such activity we generated a minimal construct that comprises much of the 23S rRNA peptidyl transferase
center, including the central loop and the A- and P-loops. This minimal rRNA domain was inactive in peptide bond formation
under all conditions tested. The RNA was subsequently subjected to six rounds of in vitro selection designed to enrich for
this activity. The result was a mutated rRNA sequence that could catalyze the covalent linkage of an A-site and P-site substrate;
however, the product did not contain a peptide bond. The current study is an example of an in vitro derived alternate function
of rRNA mutants and illustrates the evolutionary possibility that the protoribosome may have used amino acids as substrates
before it gained the ability to join them into peptides. Though peptidyl transferase activity in the absence of protein remains
elusive, the ease with which alternate catalytic activity was selected from rRNA with a small number of mutations suggests
that rRNA may have inherent activity. This study represents a step on the path toward isolating that native activity.
Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
[Reviewing Editor: Dr. Niles Lehman] 相似文献
16.
Chotimarkorn C Nagasaka R Ushio H Ohshima T Matsunaga S 《Biochemical and biophysical research communications》2005,338(2):1222-1228
A novel fluorescent probe 3-perylene diphenylphosphine (3-PeDPP) was synthesized for the direct analysis of lipid hydroperoxides. The structure of 3-PeDPP was identified by the spectroscopic data, FAB-MS, (1)H NMR, and (13)C NMR. The reactivities of 3-PeDPP with lipid hydroperoxides were investigated in chloroform/MeOH homogeneous solutions and PC liposome model systems oxidized by either 2,2'-azobis(2-amidinopropane)dihydrochloride and photosensitized oxidation. The fluorescence intensity derived from 3-perylene diphenylphosphineoxide (3-PeDPPO) increased proportionally with amount of hydroperoxides produced in homogeneous solutions and liposome model systems. 3-PeDPP was easily incorporated into mouse myeloma SP2 cells and thin tissue section for dynamic membrane lipid peroxidation studies. Linear correlations between fluorescence intensity and amount of hydroperoxides in the cell membrane and tissue sections were obtained. The fluorescence intensity from 2-dimensional image analysis was also well correlated with lipid hydroperoxide level in these models. Thus, the novel probe 3-PeDPP is useful for the direct determination of lipid hydroperoxides in biological materials. 相似文献
17.
In vitro selection of high-affinity nucleic acid ligands to parasite target molecules 总被引:2,自引:0,他引:2
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. 相似文献
18.
C. Stewart Gillespie Lise Bernier Peter J. Brophy David R. Colman 《Journal of neurochemistry》1990,54(2):656-661
We have investigated the site of synthesis of the 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs I and II) in rat brain. Rapid kinetics of incorporation of CNPs into oligodendrocyte plasma membrane in the intact brain are consistent with their synthesis on free polysomes. This hypothesis was confirmed by the translation in vitro of RNA isolated from free and bound polysomes, respectively. Unlike myelin basic protein (MBP) mRNAs, CNP mRNAs are not enriched in a myelin-associated pool of RNA. MBPs, but not CNPs, were found to readily associate in vitro with membrane vesicles derived from rough endoplasmic reticulum. The avidity of MBPs in binding to membranes is probably related to the previously observed spatial segregation of MBP mRNAs into actively myelinating cellular processes of the oligodendrocyte. Such a segregation would ensure that newly synthesized MBPs are immediately incorporated into myelin. In contrast, the CNPs probably associate with the cytoplasmic surface of the oligodendrocyte plasma membrane through interaction with a membrane-bound receptor. 相似文献
19.
Mitsuru Endo Yuki HasegawaSeiji Fukuda Hironori KobayashiYuka Yotsumoto Yuichi MushimotoHong Li Jamiyan PurevsurenSeiji Yamaguchi 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(20):1673-1676
Glutaric aciduria type 2 (multiple acyl-CoA dehydrogenase deficiency, MAD) is a multiple defect of mitochondrial acyl-CoA dehydrogenases due to a deficiency of electron transfer flavoprotein (ETF) or ETF dehydrogenase. The clinical spectrum are relatively wide from the neonatal onset, severe form (MAD-S) to the late-onset, milder form (MAD-M). In the present study, we determined whether the in vitro probe acylcarnitine assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry (MS/MS) can evaluate their clinical severity or not. Incubation of cells from MAD-S patients with palmitic acid showed large increase in palmitoylcarnitine (C16), whereas the downstream acylcarnitines; C14, C12, C10 or C8 as well as C2, were extremely low. In contrast, accumulation of C16 was smaller while the amount of downstream metabolites was higher in fibroblasts from MAD-M compared to MAD-S. The ratio of C16/C14, C16/C12, or C16/C10, in the culture medium was significantly higher in MAD-S compared with that in MAD-M. Loading octanoic acid or myristic acid led to a significant elevation in C8 or C12, respectively in MAD-S, while their effects were less pronounced in MAD-M. In conclusion, it is possible to distinguish MAD-S and MAD-M by in vitro probe acylcarnitine profiling assay with various fatty acids as substrates. This strategy may be applicable for other metabolic disorders. 相似文献
20.
Baritaki S Zafiropoulos A Georgopoulos E Souris S Krambovitis E 《Cancer immunology, immunotherapy : CII》2001,50(2):109-114
It has been demonstrated that IgG antibodies can be generated to self-antigen peptides as well as against viral antigens
by an antigen-specific in vitro immunization system of resting human peripheral B-lymphocytes. Using a synthetic peptide from
the consensus variable tandem-repeat region of the MUC3 mucin (TSSITTTGTTSHSTPSP) as the B cell epitope, we immunized blood
donor B-lymphocytes in vitro and tested for MUC3-specific antibodies by ELISA. After the primary activation step all antibodies
were IgM. At the end of the secondary immunization step we obtained 1.8% (21/1138) of the cultures with IgG-switched antibodies.
In a competitive inhibition ELISA using the MUC1, MUC2, MUC3, MUC4 and PIP2 peptides, only one culture (F8.1) gave satisfactory
specific inhibition. Using this antibody in fluorometric studies, it stained cells from two colon carcinoma cell lines predominantly
in the cytoplasm, whereas those from a breast cancer cell line stained predominantly the cell surface. In a preliminary immunohistological
evaluation with formalin-fixed sections, the antibody appeared to moderately stain colon sections, but not breast sections
or lymph node. This method of in vitro immunization may be a useful tool in generating IgG antibodies specific to self-antigens
and could find applications in tumour targeting and immunotherapy.
Received: 12 October 2000 / Accepted: 11 January 2001 相似文献