Specific DNA Aptamer Research Articles

Highly sensitive determination of recombinant human erythropoietin-α in aptamer-based affinity probe capillary electrophoresis with laser-induced fluorescence detection

Recombinant human erythropoietin-α (rHuEPO-α) has been widely used in clinic for anemia treatment. The detection and quantification of rHuEPO-α is essential for monitoring this widespread recombinant glycoprotein pharmaceutical. In this paper, we developed a new affinity probe capillary electrophoresis/laser-induced fluorescence (APCE/LIF) method for the detection of rHuEPO-α by using a specific single-stranded DNA aptamer probe for the first time. In this method, the complex of aptamer-rHuEPO-α and the free aptamer can be well separated and identified by their migration and fluorescence intensity after systematic optimization. The existence of sodium cation in the sample buffer and running buffer played a critical role for stabilizing complex and enhancing the separation efficiency, additionally, suitable high voltage and sample buffer additives were also important for improving the peak height of the complex. Under the optimized conditions, the method was successfully applied for the quantification of rHuEPO-α in physiological buffer, artificial urine and human serum. The linear range for rHuEPO-α was from 0.2 to 100 nM and the limit of detection was 0.2 nM (i.e. 7.4 ng/mL). Further binding experiments using fluorescein isothiocyanate-labeled rHuEPO-α (F-rHuEPO-α) and N-deglycosylated F-rHuEPO-α demonstrated that the oligosaccharides moiety was of importance in the specific interaction between rHuEPO-α and its aptamer.

In vivo endothelialization of heart valves using high specific DNA-aptamers as capture molecules for circulating endothelial progenitor cells (EPCs)

In vivo endothelialization of heart valves using high specific DNA-aptamers as capture molecules for circulating endothelial progenitor cells (EPCs)

Aptamer-Based Regionally Protected PCR for Protein Detection

DNA aptamers are single-stranded nucleotide sequences that bind specifically to target molecules. By combining the advantages of PCR for amplifying specific DNA sequences and aptamer technology, we have developed a new strategy to detect target molecules such as proteins. Ovine follicle-stimulating hormone alpha subunit (oFSHalpha) was used as the model protein to generate a specific DNA aptamer via an in vitro evolutionary process. A targeted regional-mapping approach and a target-capturing assay were used to identify the binding region on the aptamer molecule. In the detection assay, referred to as "aptamer-based regionally protected PCR" (ARP-PCR), the aptamer was allowed to bind to the target protein in solution before digestion with DNase I. The region of the aptamer bound to the target was protected from DNase I cleavage. The target-binding region of the aptamer protected from the enzymatic treatment was then amplified by the PCR. Aptamers against oFSHalpha were generated. Six sequences of 20 selected aptamer clones were identical. This aptamer sequence was divided into 4 regions according to the aptamer's secondary structure. From examination of the target-binding ability of each region, we determined the specific binding region, for which primers were designed. With the aptamer and primers to detect oFSHalpha by means of the ARP-PCR method, we were able to detect the target protein at concentrations as low as 10(-14) mol/L. Combining the use of a DNA aptamer with the PCR is a potentially useful analytic tool for detection of proteins at low concentrations. .

In vivo Tissue Engineering of Heart Valves: Evolution of a Novel Concept

Current tissue-engineering principles of heart valves include tissue- or stem cell-derived cells with subsequent in vitro incubation on various scaffolds prior to implantation. Limitations of this approach include a long in vitro culture, an accompanied risk of infection and sophisticated, cost-intensive infrastructures. An 'off-the-shelf' heart valve with in vivo endothelialization and tissue-regeneration potential would overcome these limitations. Additionally, the development of a heart valve with growth potential would be a huge improvement for pediatric patients. This article discusses different starter matrices, homing and immobilization strategies of host cells and masking approaches of inflammatory structures for in vivo surface and tissue engineering of heart valves. Novel concepts will be presented based on highly specific DNA-aptamers immobilized on the heart valve surface as capture molecules for endothelial progenitor cells circulating in the bloodstream.

Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells

We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

Selection and characterization of DNA aptamers against VEGF165 with aptamer blotting method and its application

We have isolated two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF165). To select aptamers that have high specificity for VEGF165, we used another protein as a competitor in the SELEX process. In addition, we evaluated the screened library with aptamer blotting. Isolated aptamers bound to VEGF165 with Kd in a few hundred-nanomolar ranges, and did not bind to a competitor. This selection method allowed us to select the specific aptamers against a target protein efficiently. Furthermore, isolated aptamers did not bind to VEGF121. These specific DNA aptamers against VEGF165 would be a useful sensing element for cancer diagnosis.

Single-chain antibodies against DNA aptamers for use as adapter molecules on DNA tile arrays in nanoscale materials organization

Complex DNA nanostructures have been developed as structural components for the construction of nanoscale objects. Recent advances have enabled self-assembly of organized DNA nanolattices and their use in patterning functional bio-macromolecules and other nanomaterials. Adapter molecules that bind specifically to both DNA lattices and nanomaterials would be useful components in a molecular construction kit for patterned nanodevices. Herein we describe the selection from phage display libraries of single-chain antibodies (scFv) for binding to a specific DNA aptamer and their development as adapter molecules for nanoscale construction. We demonstrate the decoration of various DNA tile structures with aptamers and show binding of the selected single-chain antibody as well as the self-assembly of mixed DNA-protein biomolecular lattices.

Editing by a tRNA synthetase: DNA aptamer-induced translocation and hydrolysis of a misactivated amino acid.

Aminoacyl-tRNA synthetases establish the rules of the genetic code by aminoacylation reactions. Occasional activation of the wrong amino acid can lead to errors of protein synthesis. For isoleucyl-tRNA synthetase, these errors are reduced by tRNA-dependent hydrolytic editing reactions that occur at a site 25 A from the active site. These reactions require that the misactivated amino acid be translocated from the active site to the center for editing. One mechanism describes translocation as requiring the mischarging of tRNA followed by a conformational change in the tRNA that moves the amino acid from one site to the other. Here a specific DNA aptamer is investigated. The aptamer can stimulate amino acid-specific editing but cannot be aminoacylated. Although the aptamer could in principle stimulate hydrolysis of a misactivated amino acid by an idiosyncratic mechanism, the aptamer is shown here to induce translocation and hydrolysis of misactivated aminoacyl adenylate at the same site as that seen with the tRNA cofactor. Thus, translocation to the site for editing does not require joining of the amino acid to the nucleic acid. Further experiments demonstrated that aptamer-induced editing is sensitive to aptamer sequence and that the aptamer is directed to a site other than the active site or tRNA binding site of the enzyme.

DNA aptamer targets translational editing motif in a tRNA synthetase

Potential errors in translation occur when the wrong amino acid is activated by an aminoacyl tRNA synthetase to form a misactivated aminoacyladenylate. The misactivated amino acid can in the next step be attached to a rRNA that has an anticodon different than the ones corresponding to the amino acid. If the misacylated tRNA donates its amino acid to a growing polypeptide chain, then an error of translation occurs. However, certain tRNA synthetases have an editing activity that corrects errors of misactivation and of misacylation. The relationship between these two error-correcting activities in a synthetase has not been clear. We showed recently that an insertion (known as CPI) into the active site of a class I tRNA synthetases has a deacylase activity that hydrolytically removes mischarged amino acids that are attached to tRNAs. In other work, we showed that a specific DNA aptamer, selected from a random pool, could stimulate hydrolytic breakdown of a misactivated aminoacyladenylate bound to a tRNA synthetase. In this work, we photo-crosslinked the DNA aptamer to the tRNA synthetase. A single crosslinked peptide on the synthetase was identified. This peptide is located within the CPI insertion, adjacent to residues known to affect the amino acid specificity of the tRNA deacylase activity. These results raise the possibility that the CPI insertion has a role not only in correcting misacylations, but also in the hydrolytic breakdown of misactivated aminoacyladenylates.