共查询到20条相似文献,搜索用时 31 毫秒
1.
We have developed an aptameric enzyme subunit (AES) for immunoglobulin E (IgE) sensing. AES is an artificial enzyme subunit
constructed from two different aptamers and does not require any modification. Using the AES, the target molecule can be detected
by measuring enzymatic activity in homogeneous solution. We connected IgE-binding aptamer and its complementary strand to
split thrombin-inhibiting aptamer. The hybrid of these two oligonucleotides inhibited thrombin activity and it decreased in
the presence of IgE. We were able to detect IgE by using this AES in homogeneous solution with a detection limit of 50 pmol. 相似文献
2.
Yoshida W Mochizuki E Takase M Hasegawa H Morita Y Yamazaki H Sode K Ikebukuro K 《Biosensors & bioelectronics》2009,24(5):1116-1120
We selected DNA aptamers against insulin and developed an aptameric enzyme subunit (AES) for insulin sensing. The insulin-binding aptamers were identified from a single-strand DNA library which was expected to form various kinds of G-quartet structures. In vitro selection was carried out by means of aptamer blotting, which visualizes the oligonucleotides binding to the target protein at each round. After the 6th round of selection, insulin-binding aptamers were identified. These identified insulin-binding aptamers had a higher binding ability than the insulin-linked polymorphic region (ILPR) oligonucleotide, which can be called a "natural" insulin-binding DNA aptamer. The circular-dichroism (CD) spectrum measurement of the identified insulin-binding DNA aptamers indicated that the aptamers would fold into a G-quartet structure. We also developed an AES by connecting the best identified insulin-binding aptamer with the thrombin-inhibiting aptamer. Using this AES, we were able to detect insulin by measuring the thrombin enzymatic activity without bound/free separation. 相似文献
3.
We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA–enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein. A hexahistidine tag was genetically attached to the C terminus of the GST, and the 5′ end of an oligonucleotide was conjugated with nitrilotriacetic acid (NTA) for the site-specific conjugation of the DNA with the GST based on a Ni2+ complex interaction. We found that fluorescein acted as a weak inhibitor of GST and succeeded in the regulation of GST activity by increasing the local concentration of the weak inhibitor by the hybridization of a 3′-end fluorescein-modified DNA. The catalytic activity of the DNA aptamer–enzyme conjugate showed a dose-dependent response to thrombin, indicating that the GST activity was clearly recovered by the binding of the DNA aptamer to thrombin. The current system enables the sensitive and specific detection of thrombin simply by measuring the enzymatic activity in a homogeneous medium. 相似文献
4.
We previously identified a thrombin-inhibiting DNA aptamer that was presumed to form a G-quartet structure with a duplex. To investigate the importance of the sequences in the duplex region and to obtain aptamers with higher inhibitory activities, we randomized the sequences of the duplex region of this aptamer and carried out selection based on inhibitory activity using a genetic algorithm. This method consisted of selection via an inhibition assay, crossover, and mutation in silico. After two cycles, we obtained ligands with greater inhibitory activities than that of the original aptamer. In addition, the duplex sequences were found to contribute to the inhibitory activities of aptamers. 相似文献
5.
Kubiński K Domańska K Sajnaga E Mazur E Zieliński R Szyszka R 《Molecular and cellular biochemistry》2007,295(1-2):229-236
Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears
to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic
(α and/or α′) subunits and two regulatory β and β′ subunits. Previously, we have reported isolation from yeast cells four
active forms of CK2, composed of αα′ββ′, α2ββ′, α′2ββ′ and a free α′-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory β subunit cannot substitute other β′ subunit and only both of them can form fully active enzymatic
unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing
single deletion of the β or β′ regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation
experiments show that polyadenylation factor Fip1 interacts with catalytic α subunits of CK2 and interaction with beta subunits
in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast
protein kinase CK2β/β′ subunits in the regulation of holoenzyme assembly and phosphotransferase activity. 相似文献
6.
This report describes a novel and efficient method for walking the sequence of a genomic deoxyribonucleic acid (DNA) from
a known region to an unknown region based on an oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction technique.
In this method, genomic DNA is digested by a restriction enzyme that generates a sticky 5′-end, followed by ligation of a
one-base excess oligo-adaptor using T4 DNA ligase. The adaptor consists of two complementary oligos that form the same sticky
end as the digested genomic DNA fragments, except that the 5′-overhang base overlaps the corresponding 3′-end base of the
restriction site. This overhanging terminal base prevents ligation between the adaptors, and the appropriate molar ratio of
adaptor to genomic DNA enables specific amplification of the target sequence. T4 DNA ligase catalyzes both the ligation of
the phosphorylated overhang base of the adaptor to genomic DNA and the excision of the corresponding 3′-terminal base of the
genomic DNA. This sequence-specific exonuclease activity of T4 DNA ligase was confirmed by ligation of an alternative adaptor
in which the 5′-terminal base was not consistent with the corresponding 3′-terminal base. Using this technique, the 3′- and
5′-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined. 相似文献
7.
P. H. Benetti S. I. Kim M. Canonge T. Chardot J. C. Meunier 《Molecular & general genetics : MGG》1997,256(4):355-364
Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form αα′β2. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic α subunit
of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron
was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity
with those of α subunit described in other species, although, uniquely, Y. lipolytica CKIIα lacks cysteines. We find that the α subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKIIα expression and CKIIα activity. We show that expression of this enzyme is regulated. The catalytic
subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts.
Received: 20 December1997 / Accepted: 19 June 1997 相似文献
8.
G. A. Hitman G. C. Toms B. J. Boucher L. Garde P. Baker J. Awad H. Festenstein 《Immunogenetics》1989,30(6):427-431
Cytokines and their related enzyme pathways may play a part in the development of insulin-dependent diabetes mellitus (IDDM).
We have therefore studied the activity of the enzyme 2′–5′ oligoadenylate synthetase (which is induced by both interferon
and the tumour necrosis factors) in circulating mononuclear cells from 40 subjects with IDDM and 32 healthy control subjects.
There was no difference in mean basal enzyme activity between the two groups. A polymorphism of the 2′–5′ oligoadenylate synthetase
gene, not previously described, was found using the restriction enzymeBam HI. There was no association of 2′–5′ oligoadenylate synthetase genotypes with IDDM, but there was a significant correlation
between basal 2′–5′ oligoadenylate synthetase activity and 2′–5′ oligoadenylate synthetase genotypes. Significantly higher
mean basal levels of 2′–5′ oligoadenylate synthetase activity were associated with HLA-DQA 4.6 phenotype (determined using
the restriction enzymeTaq 1 and a DQA probe) and HLA-DR3 (determined serologically), whereas significantly lower mean levels of enzyme activity were
associated with HLA-DQA 5.5 and HLA-DR7, in both IDDM and control subjects. An analysis of variance confirmed that these associations
were independent 2′–5′ oligoadenylate synthetase genotype. Likewise, a significantly higher mean level of enzyme activity
was associated with the heterozygous 1/3 insulin-related genotype in the IDDM subjects only. This study therefore suggests
that the possession of certainHLA haplotypes might be associated with differing levels of basal 2′–5′ oligoadenylate synthetase activity. 相似文献
9.
Arvind Misra Pratibha Dwivedi Mohammad Shahid 《Russian Journal of Bioorganic Chemistry》2009,35(1):62-67
A modified fluorescent probe UFAA AAT CTC CGC CGC was synthesized using the nucleoside analogue 3′-O-(N,N′-diisopropylamino-2-cyanoethoxyphosphinyl)-5′-O-(4,4′-dimethoxytrityl)-2′-O-(dansyl-1-sulfonamidohexylaminocarbonyl)uridine for hybridization studies with perfectly matched (U/A) complementary DNA
and with a DNA strand having similar G-rich telomeric units at their 3′-ends. Data on the thermal stability and decrease in
fluorescence intensity due to the presence of dG units clearly demonstrated the potential application of this approach in
DNA diagnostics in homogeneous hybridization assays.
The text was submitted by the authors in English. 相似文献
10.
Fred W. Perrino Dan J. Mazur Heather Ward Scott Harvey 《Cell biochemistry and biophysics》1999,30(3):331-352
The polymerization of nucleotide analogs into DNA is a common strategy used to inhibit DNA synthesis in rapidly dividing tumor
cells and viruses. The mammalian DNA polymerases catalyze the insertion of the arabinofuranosyl analogs of dNTPs (aranucleotides)
into DNA efficiently, but elongate from the 3′ aranucleotides poorly. Slow elongation provides an opportunity for exonucleases
to remove aranucleotides. The exonuclease activity associated with DNA polymerase δ removes araCMP from 3′ termini with the
same efficiency that it removes a paired 3′ deoxycytosine suggesting that the proofreading exonucleases associated with DNA
polymerases might remove aranucleotides inefficiently. A separate 30 kDa exonuclease has been purified from mammalian cells
that removes araCMP from 3′ termini. The activity of this enzyme in the cell could remove aranucleotides from 3′ termini of
DNA and decrease the efficacy of the analogs. Inhibition analysis of the purified exonuclease shows that this enzyme is inhibited
by thioinosine monophosphate (TIMP) with aK
i=17 μM. When high TIMP levels are generated in HL-60 cells, incorporation of araC in DNA is increased about 16-fold relative to
total DNA synthesis. This increased araC in DNA is likely a result of exonuclease inhibition in the cell. Thus, exonucleases
in cells might play an important role in removing aranucleotides inserted by DNA polymerases. 相似文献
11.
R. J. Phillips D. C. Hickleton P. E. Boehmer P. T. Emmerson 《Molecular & general genetics : MGG》1997,254(3):319-329
To investigate the role that the individual subunits play in the ATP-dependent helicase activity of the RecBCD protein we
have investigated the ability of the RecB, RecC and RecD proteins to displace various 20-mer oligonucleotides annealed to
either end or to the centre of an oligonucleotide 60 bases long. The results show that the only subunit which can displace
the 20-mers in the absence of the other subunits is the RecB protein. Moreover, the 20-mer is displaced only if it is annealed
to the 60-mer at the 5′ end or the middle, suggesting that the RecB protein translocates along the 60-mer in the 3′ to 5′
direction, displacing annealed 20-mers as it proceeds. We have shown that reconstituted RecBC and RecBCD complexes displace
the 20-mers but, unlike RecB, they do not require a 3′-ended single-stranded region for helicase action, but can displace
the 20-mers from either end of the 60-mer. The level of helicase activity of the RecBC complex is considerably greater than
that of RecB alone, and the activity of the RecBCD complex appears to be greater still. This hierarchy of activity is also
shown by DNA binding studies, but is not reflected in the ATPase activities of the enzymes. We have also shown that the ability
of trypsin to cleave various sites on the RecB molecule is modified by the presence of ATP or ATP-γ-S, suggesting that conformational
changes may be induced in RecB upon ATP binding. We discuss a model for the ATP-driven, unidirectional motion of the RecB
translocase along single-stranded DNA. In this model, the RecB molecule binds to single-stranded DNA and then translocates
along it, one base at a time, in the 3′ to 5′ direction, by a `ratchet' mechanism in which repeated stretching and contraction
of the protein is coupled to ATP hydrolysis. The RecC protein in the RecBC complex is proposed to act as a `sliding clamp'
which increases processivity by preventing dissociation.
Received: 10 September 1996 / Accepted: 18 November 1996 相似文献
12.
Takashi Sakamoto Akio Kobori Masako Shigezawa Yasutaka Amitani Maiko Higuchi Akira Murakami 《Nucleosides, nucleotides & nucleic acids》2013,32(10-12):1659-1664
We developed a bispyrene-conjugated 2 ′-O-methyloligoribonucleotide as an RNA-specific RNA-probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and discriminating RNA from DNA. The assay was carried out in homogeneous aqueous media without removing the unbound probe from the detection solution. This homogeneous fluorescence assay also discriminated mismatch sequences in the target RNA. These pyrene probes could possess high potential to detect RNA in biological specimens simply. 相似文献
13.
Ogasawara D Hachiya NS Kaneko K Sode K Ikebukuro K 《Biosensors & bioelectronics》2009,24(5):1372-1376
Aptamers are good molecular recognition elements for biosensors. Especially, their conformational change, which is induced by the binding to the target molecule, enables the development of several types of useful detection systems. We applied this property to bound/free separation, which is a crucial process for highly sensitive detection. We designed aptamers which change their conformation upon binding to the target molecule and thereby expose a single-strand bearing the complementary sequence to the capture probe immobilized onto the support. We named the designed aptamers "capturable aptamers" and the capture probe "capture DNA". Three capturable aptamers were designed based on the PrP aptamer, which binds to prion protein. One of these capturable aptamers was demonstrated to recognize prion protein and change its conformation upon binding to it. A detection system using this designed capturable aptamer for prion protein was developed. Capturable aptamers and capture DNA allow us to perform simple bound/free separation with only one target ligand. 相似文献
14.
Sakamoto T Kobori A Shigezawa M Amitani Y Higuchi M Murakami A 《Nucleosides, nucleotides & nucleic acids》2007,26(10-12):1659-1664
We developed a bispyrene-conjugated 2'-O-methyloligoribonucleotide as an RNA-specific RNA-probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and discriminating RNA from DNA. The assay was carried out in homogeneous aqueous media without removing the unbound probe from the detection solution. This homogeneous fluorescence assay also discriminated mismatch sequences in the target RNA. These pyrene probes could possess high potential to detect RNA in biological specimens simply. 相似文献
15.
Lahja Uitto Jussi Halleeen P?ivi Remes Tapio Kesti Juhani E. Syv?oja 《Chromosoma》1992,102(Z1):S142-S146
The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes
mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide
non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary
regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to
those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA
is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a
non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition
to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase
epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity. 相似文献
16.
17.
Kaori Tsukakoshi Ryuichi Harada Koji Sode Kazunori Ikebukuro 《Biotechnology letters》2010,32(5):643-648
α-Synuclein is a native, unfolded protein that causes several neurodegenerative diseases such as dementia with Lewy bodies
and Parkinson’s disease. We have now identified the first DNA aptamers against α-synuclein using native PAGE applied to the
SELEX method. We call this aptamer “M5-15”; it is the α-synuclein-bound aptamer and was isolated after four cycles of screening.
M5-15 is composed of three stem-loop structures that may play an important role in the binding to α-synuclein. Moreover, M5-15
specifically binds to the α-synuclein monomer and oligomer. We expect that this aptamer will become a useful tool in α-synuclein
analysis and diagnosis. 相似文献
18.
Bo Feng Xueli An Zhibin Xu Dongcheng Liu Aiming Zhang Ning Wu Tao Wang 《Molecular breeding : new strategies in plant improvement》2011,28(2):163-170
A novel chimeric high-molecular-weight (HMW) glutenin subunit gene from a new common wheat line W958 (2n = 6x = 42) was isolated and characterized. SDS–PAGE analysis revealed that this glutenin subunit has similar electrophoretic mobility
to 1Dx5, so it was designated 1Dx5′. Genomic DNA from W958 was amplified and a 2,505-bp fragment was obtained. The 1Dx5′ subunit
showed a chimeric primary structure of 1Dx5 and 1Dx2, with the 1Dx5 sequence in the 5′ and middle repetitive regions and the
1Dx2 sequence in the repetitive domain and 3′ region. MALDI-TOF-MS analysis demonstrated that 1Dx5′ had a molecular weight
of 86815.1 Da, close to that of an x-type glutenin subunit. Secondary structure analysis showed that this subunit had six
helixes and one strand, including four helixes in the repetitive domain which could enhance the dough properties. Additionally,
the promoter of 1Dx5′ was obtained and showed the same sequence as 1Dx5 or 1Dx2 except for a few base conversions. The promoter analysis indicated that the cis-acting regulatory elements of 1Dx5′ were the same as those of 1Dx5 and/or 1Dx2. Previously, we have demonstrated that this novel glutenin subunit is associated with good bread-making quality and comprises
a very large proportion of the F2 segregation population. Consequently, we suggest that the amino acid residue composition
and the secondary structure of the subunit may contribute to the bread-making quality. In summary, the novel 1Dx5′ gene could have greater potential in wheat quality improvement. 相似文献
19.
Huiyu Liang Yusheng Shi Zhewen Kou Yonghua Peng Wenjun Chen Xiaowen Li Shuji Li Ying Wang Fang Wang Xingmei Zhang 《PloS one》2015,10(10)
An initial step in amyloid-β (Aβ) production includes amyloid precursor protein (APP) cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer’s disease (AD). Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX). A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity. 相似文献