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ABSTRACT: Rapid and simple methods for microRNA (miRNA) detection are essential for biological research of miRNAs and clinical diagnosis. Here we describe a sensitive and specific real time RT-PCR (also RT-qPCR) method for miRNA quantification. The whole detection process including reverse transcription and PCR is performed in one PCR tube by a one-step operation on a real-time PCR system. The results display a wide linear range from 0.1amol to 10fmol with a detection limit of 12.6zmol for miRNA let-7a detection. Let-7a in small RNA samples extracted from tumor cells has been successfully detected by this method. This method is cost-effective, simple and rapid, and has the advantages in the high-throughput routing assay of given miRNAs, as well as in non-model research that has less specific kits and reagents.
Talanta 06/2013; 110:190-5. · 3.79 Impact Factor
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ABSTRACT: G-quadruplexes (G4s) are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs) are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes) have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s.
PLoS ONE 01/2013; 8(4):e62348. · 4.09 Impact Factor
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ABSTRACT: In this paper, we describe a turn-on fluorescent probe for Zn(2+) and Cd(2+) ion detection and discrimination. This probe, N'-bis-(N,N-di-(2-pyridylmethyl)-ethane-1,2-diamine)-perylene-3,4,9,10-tetracarboxylic-diimide (PDI-DIDPA), exhibits very low fluorescence at pH above 6.0 due to the photo-induced electron transfer (PET) process from 2-pyridylmethyl-amine (DPA) to perylene tetracarboxylic diimide (PDI). Zn(2+) and Cd(2+) can chelate PDI-DIDPA to form a stable complex at pH 6.0-7.0 and 9.0 respectively, and inhibit the PET process, which result in the fluorescence recovery of PDI-DIDPA. This fluorescence turn-on behavior allows the detection of Zn(2+) in the range of 0.1-4.0 μM and Cd(2+) in the range of 0.1-5.0 μM. The limit of detection for Zn(2+) and Cd(2+) is as low as 32 nM and 48 nM. The high selectivity, high sensitivity and easy operation make this probe suitable for the rapid detection of Zn(2+) and Cd(2+) respectively. The different response of PDI-DIDPA to Zn(2+) and Cd(2+) at different pH makes it possible to discriminate Zn(2+) and Cd(2+) by simply adjusting the pH of the working solution.
The Analyst 12/2012; · 4.23 Impact Factor
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ABSTRACT: Through substituting two canonical base pairs of a streptavidin-binding aptamer with T-T mismatched base pairs, a new aptamer was constructed. Its binding ability could be controlled by Hg(2+) through the formation of T-Hg-T metal-base pairs.
Chemical Communications 11/2012; · 6.17 Impact Factor
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ABSTRACT: The screening of ligands against proteins plays important role in drug discovery and biological research. Using a dye labelled Streptavidin binding aptamer (SBA) as a competitive reporter probe, we found that adenosine bound to streptavidin specifically. Fluorescence spectral analysis showed that adenosine bound to both avidin and streptavidin with the K(ds) in the range of 0.1-0.2mM, and these bindings can be blocked by biotin. Although streptavidin and avidin are well-known and widely used in bioanalysis, their biological role is still a riddle so far. Since adenosine is a ubiquitous physiological regulator present in cells, our finding provides new clues for the understanding of the functions of both proteins.
Bioorganic & medicinal chemistry letters 10/2012; · 2.65 Impact Factor
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ABSTRACT: Zeatins, a major type of cytokinin, are ubiquitous in higher plants, and involve in regulating a wide range of developmental processes. The development of highly specific ligands to zeatins would be very useful in plant biological research. Here we describe a group of oligonucleotide ligands (aptamers) generated against trans-zeatin. The optimized aptamers possess strong affinity to trans-zeatin and trans-zeatin riboside (Kd=3-5μM), and relatively weak affinity (Kd=27-30μM) to cis-zeatin and dihydrozeatin. These aptamers adopt a hairpin-G-quadruplex structure for binding to zeatin. A fluorescence turn-on aptasensor based on graphene oxide (GO) was developed for the recognition of zeatins. The specificity assay of this aptasensor shows good response to zeatins, and no response to the adenine derivatives (analog of zeatins) abundantly existing in biological samples. These results show the great potential of these aptamers in chemical analysis and biological investigation of zeatins.
Biosensors & bioelectronics 08/2012; · 5.43 Impact Factor
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ABSTRACT: Aptamers are usually generated against a specific molecule. Their high selectivity makes them only suitable for studying specific targets. Since it is nearly impossible to generate aptamers for every molecule, it can be of great interest to select aptamers recognizing a common feature of a group of molecules in many applications. In this paper, we describe the selection of aptamers for indirect recognition of alkyl amino groups. Because amino groups are small and positive charged, we introduced a protection group, p-nitrobenzene sulfonyl (p-nosyl) to convert them into a form suitable for aptamer selection. Taking N(ε)-p-nosyl-l-lysine (PSL) as a target, we obtained a group of aptamers using the SELEX technique. Two optimized aptamers, M6b-M14 and M13a exhibit strong affinity to PSL with the K(d) values in the range of 2-5 μM. They also show strong affinity to other compounds containing p-nosyl-protected amino groups except those also possessing an α-carboxyl group. Both aptamers adopt an antiparallel G-quadruplex structure when binding to targets. An aptamer beacon based on M6b-M14 showed good selectivity toward the reaction mixture of p-nosyl-Cl and alkyl amino compounds, and could recognize lysine from amino acid mixtures indirectly, suggesting that aptamers against a common moiety of a certain type of molecules can potentially lead to many new applications. Through this study, we have demonstrated the ability to select aptamers for a specific part of an organic compound, and the chemical conversion approach may prove to be valuable for aptamer selection against molecules that are generally difficult for SELEX.
Analytical Chemistry 08/2012; 84(17):7323-9. · 5.86 Impact Factor
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ABSTRACT: G-quadruplex (G4) is a characteristic secondary structure of nucleic acids containing repetitive tandem guanines. G4-forming sequences are found prevalent in the human genome by bioinformatics analysis. Accumulating evidence has suggested that G4s are involved in many biological processes. Selective isolation of G4s would be an effective tool in the study of G4s. In this paper, we prepared four affinity matrixes using hemin or a perylene derivative (N,N'-Bis-(2-(amino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide, Pery01) as ligand, and investigated the retention behaviors of different G4s on these matrixes. Our experimental results suggest that the π-π stacking interaction between ligand and G-tetrad plays a key role in the selective isolation of G4s, whereas the electrostatic interaction between DNA and matrix causes the nonspecific binding. One matrix prepared by immobilizing Pery01 on polyglycidylmethacrylate (PGMA) beads through an aminocaproic acid spacer exhibits good selectivity for parallel structure G4s and has been successfully used to directly isolate a spiked parallel G4 from plasma.
Journal of chromatography. A 02/2012; 1246:62-8. · 4.19 Impact Factor
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ABSTRACT: Recently, G-quadruplex/hemin (G4/hemin) complexes have been found to exhibit peroxidase activity, and this feature has been extensively exploited for colorimetric detection of various targets. To further understand and characterize this important DNAzyme, its substrate specificity, inactivation mechanism, and kinetics have been examined by comparison with horseradish peroxidase (HRP). G4/hemin DNAzyme exhibits broader substrate specificity and much higher inactivation rate than HRP because of the exposure of the catalytic hemin center. The inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H(2)O(2) rather than the destruction of G4. Both the inactivation rate and catalytic oxidation rate of G4/hemin DNAzyme depend on the concentration of H(2)O(2), which suggests that active intermediates formed by G4/hemin and H(2)O(2) are the branch point of catalysis and inactivation. Reducing substrates greatly inhibit the inactivation of G4/hemin DNAzyme by rapidly reacting with the active intermediates. A possible catalytic and inactivation process of G4/hemin has been proposed. These results imply a potential cause for the hemin-mediated cellular injury and provide insightful information for the future application of G4/hemin DNAzyme.
Chemistry 11/2011; 17(51):14475-84. · 5.93 Impact Factor
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ABSTRACT: Ligands specific to bioactive molecules play important roles in biomedical researches and applications, such as biological assay, diagnosis and therapy. Systemin is a peptide hormone firstly identified in plant. In this paper we report the selection of a group of DNA aptamers that can specifically bind to systemin. Through comparing the predicted secondary structures of all the aptamers, a hairpin structure with G-rich loop was determined to be the binding motif of these aptamers. The G-rich loop region of this binding motif was further characterized to fold into an antiparallel G-quadruplex by truncation-mutation assay and CD spectrum. The apparent equilibrium dissociation constant (K(d)) of one strong binding sequence (S-5-1) was measured to be 0.5 μM. The specificity assay shows that S-5-1 strongly bind to whole systemin, weakly bind to truncated or mutated systemin and does not bind to the scrambled peptide with the same amino acid composition as systemin. The high affinity and specificity make S-5-1 hold potentials to serve as a molecular ligand applied in detection, separation and functional investigation of systemin in plants.
Bioorganic & medicinal chemistry 06/2011; 19(14):4211-9. · 2.82 Impact Factor
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ABSTRACT: DNA aptamers for specific recognition of L-tryptophan have been evolved by a SELEX (systematic evolution of ligands by exponential enrichment) technique. Truncation-mutation experiments suggest that a 34-mer sequence, Trp3a-1, possesses the strongest binding ability to L-tryptophan. Trp3a-1 is predicted to adopt a loop-stem secondary structure, in which the loop may further fold into a binding pocket for L-tryptophan with the help of the stem. The specificity investigation shows that Trp3a-1 strongly binds to L-tryptophan, has almost no binding to other amino acids, and weakly binds to some tryptophan analogs and peptides containing the L-tryptophan residue. The binding of Trp3a-1 to L-tryptophan is mainly contributed to by hydrogen bonds and precise stacking formed between the binding pocket of Trp3a-1 and all groups on L-tryptophan. This aptamer has also been proved to be an effective ligand for the chiral separation of D/L-tryptophan. L-tryptophan and its derivatives are known to play important biological roles; this aptamer ligand could be used as a tool for the analysis of tryptophan and other related studies.
The Analyst 11/2010; 136(3):577-85. · 4.23 Impact Factor
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03/2010; , ISBN: 9780470571224
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ABSTRACT: Protein tyrosine kinase-7 (PTK7) is a catalytically inactive receptor tyrosine kinase (RTK). PTK7 is upregulated in many common human cancers, including colon cancer, lung cancer, gastric cancer and acute myeloid leukemia. The reason for this up-regulation is not yet known. To explore the functional role of PTK7, the expression of PTK7 in HCT 116 cells was examined using small interference (siRNA)-mediated gene silencing. Following transfection, the siRNA successfully suppressed PTK7 mRNA and protein expression. Knocking down of PTK7 in HCT 116 cells inhibited cell proliferation compared to control groups and induced apoptosis. Furthermore, this apoptosis was characterized by decreased mitochondrial membrane potential and activation of caspase-9 and -10. Addition of a caspase-10 inhibitor totally blocked this apoptosis, suggesting that caspase-10 may play a critical role in PTK7-knockdown-induced apoptosis, downstream of mitochondria. These observations may indicate a role for PTK7 in cell proliferation and cell apoptosis and may provide a potential therapeutic pathway for the treatment of a variety of cancers.
PLoS ONE 01/2010; 5(11):e14018. · 4.09 Impact Factor
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ABSTRACT: In the past two decades, high-affinity nucleic acid aptamers have been developed for a wide variety of pure molecules and complex systems such as live cells. Conceptually, aptamers are developed by an evolutionary process, whereby, as selection progresses, sequences with a certain conformation capable of binding to the target of interest emerge and dominate the pool. This protocol, cell-SELEX (systematic evolution of ligands by exponential enrichment), is a method that can generate DNA aptamers that can bind specifically to a cell type of interest. Commonly, a cancer cell line is used as the target to generate aptamers that can differentiate that cell type from other cancers or normal cells. A single-stranded DNA (ssDNA) library pool is incubated with the target cells. Nonbinding sequences are washed off and bound sequences are recovered from the cells by heating cell-DNA complexes at 95 degrees C, followed by centrifugation. The recovered pool is incubated with the control cell line to filter out the sequences that bind to common molecules on both the target and the control, leading to the enrichment of specific binders to the target. Binding sequences are amplified by PCR using fluorescein isothiocyanate-labeled sense and biotin-labeled antisense primers. This is followed by removal of antisense strands to generate an ssDNA pool for subsequent rounds of selection. The enrichment of the selected pools is monitored by flow cytometry binding assays, with selected pools having increased fluorescence compared with the unselected DNA library. The procedure, from design of oligonucleotides to enrichment of the selected pools, takes approximately 3 months.
Nature Protocol 01/2010; 5(6):1169-85. · 8.36 Impact Factor
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ABSTRACT: Here, we describe a colorimetric sensor for detecting Hg(2+) in aqueous media, which is simply constructed by the self-assembly of thymine acetamidoethanethiol (T-SH) on gold nanoparticles (AuNPs). Based on the specific interaction of Hg(2+) with two thymines (T), the T-SH modified AuNPs can be induced to aggregate through the formation of a stable T-Hg-T complex in the presence of Hg(2+), resulting in a color change from red to blue-gray. As low as 0.5 µM of Hg(2+) can be easily monitored by the naked eye using this sensor. Other metal ions, including Zn(2+), Cd(2+), Pb(2+), Ni(2+), Cu(2+), Co(2+), Mn(2+), Ba(2+), Fe(2+), Ca(2+), Mg(2+), Al(3+), and Fe(3+), could not cause any response, even at concentrations 100-fold higher than Hg(2+). The high selectivity, high stability and easy operation enable this sensor suitable for the rapid on-site detection of Hg(2+) pollution.
Analytical Sciences 01/2010; 26(11):1169-72. · 1.25 Impact Factor
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ABSTRACT: Aptamers that are selected in vitro from random pools of DNA or RNA molecules by SELEX (Systematic evolution of ligands by exponential enrichment) technique have been extensively explored for analytical and biomedical applications. Although many aptamers with high affinity and specificity against specific ligands have been reported, there is still a lack of well characterized DNA aptamers. Here we report the selection of a group of aptamer candidates (85 mer) against streptavidin. Through comparing the predicted secondary structures of all the candidates, a conservative bulge-hairpin structure section (about 29 mer) was found, and then it was determined to be the binding motif to streptavidin. This binding motif was further discovered to also exist in streptavidin-binding aptamers (SBAs) selected by three other laboratories using different methods. The primary sequences of this secondary structure motif are very different, only several nucleotides in the loop and bulge area are critical for binding and other nucleotides are variable. The streptavidin binding of all the SBAs could be competed by biotin implying that they bind to the same site on streptavidin. These results suggest that the evolution of SBA is predominated by specific groups on streptavidin. The highly variable sequence composition of streptavidin-binding aptamer would make the design of aptameric sensor or device based on streptavidin more flexible and easy.
Bioorganic & medicinal chemistry 01/2010; 18(5):1798-805. · 2.82 Impact Factor
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ABSTRACT: Here we report on the construction and evaluation of a bifunctional combined aptamer (BCA) that consists of a DNA streptavidin-binding aptamer (SBA), a DNA thrombin-binding aptamer (TBA) and a fluorophore. The BCA adopts a new conformation that is very different from simply linking the conformations of the two individual aptamers together, so that it does not bind to streptavidin in the absence of thrombin. Binding of this novel DNA aptamer to streptavidin is triggered by the thrombin binding and depends on the concentration of thrombin. Meanwhile, fluorescence from the streptavidin captured BCA reflects the quantity of the target molecule in the sample. This aptamer combination strategy based on the SBA holds good potential for applications in simultaneous detection and separation of targets of aptamers or certain DNA and RNA targets.
Biosensors & bioelectronics 11/2009; 25(6):1487-92. · 5.43 Impact Factor
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ABSTRACT: A significant number of G-quadruplex-forming sequences have been revealed in human genome by bioinformatic searches, implying that G-quadruplexes may be involved in important biological processes and may be new chemotherapeutic targets. Therefore, it is important to discover the potential interactions of G-quadruplexes with other molecules or groups. Here we describe a class of G-quadruplexes, which can bind to ethanolamine groups that widely exist in biomolecules and drug molecules. The specific interaction of these G-quadruplexes with ethanolamine groups was identified by high performance affinity chromatography (HPAC) using immobilized ethanolamine and diethanolamine as stationary phase reagents. The circular dichroism (CD) spectra and native polyacrylamide gel electrophoresis (PAGE) show that these ethanolamine binding quadruplexes adopt an intramolecularly parallel structure. The relationship of ethanolamine binding and G-quadruplexe structure provides new clues for the G-quadruplex-related studies as well as for the molecular designs of therapeutic reagents.
Biopolymers 11/2009; 91(10):874-83. · 2.87 Impact Factor
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ABSTRACT: In this work, we have developed new aptamer probes for non-small cell lung cancer (NSCLC) by directing the aptamer selection process against the living cells of adenocarcinoma, the most common subtype of NSCLC. A panel of single-stranded DNA (ssDNA) aptamers were generated and evaluated for adenocarcinoma cell recognition. The aptamers bound to the adenocarcinoma cells with dissociation constants in the nanomolar range and the binding of the selected aptamers to the adenocarcinoma cells were significantly stronger than the other cancerous lung cells as well as other types of cancer cells. Moreover, the application of the aptamers to the clinical tissue section samples showed the differentiation of adenocarcinoma from normal lung tissue and other subtypes of lung cancer. The aptamers are expected to be new molecular probes for the investigation of the molecular bases of different NSCLC subtypes and their biological heterogeneity, which is valuable for advancing NSCLC diagnosis and treatment.
The Analyst 10/2009; 134(9):1808-14. · 4.23 Impact Factor
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ABSTRACT: DNA sequences with repetitive G-rich structural motifs, which form special structures called G-quadruplexes, widely exist in the human genome. Here we report the general peroxidase activity of G-quadruplex-hemin complexes and discuss the connection between peroxidase activity and G-quadruplex structures. The high peroxidase activity of hemin complexed with intramolecular parallel G-quadruplex-forming sequences in gene promoters (such as c-Myc, VEGF, c-Kit21, HIF-1alpha, and RET) may imply a potential mechanism of hemin-mediated cellular injury. This peroxidase activity has also been demonstrated to be applicable for screening G-quadruplex ligands (potential anticancer reagents) using colorimetric and visual detection strategies.
Biochemistry 08/2009; 48(33):7817-23. · 3.42 Impact Factor
