A DNA aptamer for binding and inhibition of DNA methyltransferase 1.

DNA methyltransferases (DNMTs) are enzymes responsible for establishing and maintaining DNA methylation in cells. DNMT inhibition is actively pursued in cancer treatment, dominantly through the formation of irreversible covalent complexes between small molecular compounds and DNMTs that suffers from low efficacy and high cytotoxicity, as well as no selectivity towards different DNMTs. Herein, we discover aptamers against the maintenance DNA methyltransferase, DNMT1, by coupling Asymmetrical Flow Field-Flow Fractionation (AF4) with Systematic Evolution of Ligands by EXponential enrichment (SELEX). One of the identified aptamers, Apt. #9, contains a stem-loop structure, and can displace the hemi-methylated DNA duplex, the native substrate of DNMT1, off the protein on sub-micromolar scale, leading for effective enzymatic inhibition. Apt. #9 shows no inhibition nor binding activity towards two de novo DNMTs, DNMT3A and DNMT3B. Intriguingly, it can enter cancer cells with over-expression of DNMT1, colocalize with DNMT1 inside the nuclei, and inhibit the activity of DNMT1 in cells. This study opens the possibility of exploring the aptameric DNMT inhibitors being a new cancer therapeutic approach, by modulating DNMT activity selectively through reversible interaction. The aptamers could also be valuable tools for study of the functions of DNMTs and the related epigenetic mechanisms

Aptamer-based cocaine assay using a nanohybrid composed of ZnS/Ag2Se quantum dots, graphene oxide and gold nanoparticles as a fluorescent probe

Authors report on a new fluoro-graphene-plasmonic nanohybrid aptamer-based fluorescent nanoprobe for cocaine. To construct the nanoprobe, newly synthesized glutathione-capped ZnS/Ag 2Se quantum dots (QDs) were first conjugated to graphene oxide (GO) to form a QD-GO nanocomposite. The binding interaction resulted in a fluorescence turn-ON. Thereafter, cetyltrimethylammonium bromide (CTAB)-gold nanoparticles (AuNPs) were directly adsorbed on the QD-GO nanocomposite to form a novel QD-GO-CTAB-AuNP nanohybrid assembly that resulted in a fluorescence turn-OFF. Streptavidin (strep) was then adsorbed on the QDs-GO-CTAB-AuNP nanohybrid assembly which allowed binding to a biotinylated MNS 4.1 anticocaine DNA aptamer (B) receptor. The addition of cocaine into the strep-B-QDs-GO-CTAB-AuNP aptamer nanoprobe system aided affinity to the aptamer receptor and in turn turned on the fluorescence of the nanoprobe in a concentration-dependent manner. Under optimum experimental conditions, we found the strep-B-QD-GO-CTAB-AuNP to be far superior in its sensitivity to cocaine than the tested strep-B-QDs (no GO and CTAB-AuNPs), strep-B-QD-CTAB-AuNP (no GO) and strep-B-QD-GO (no CTAB-AuNP). In addition, the investigation of localized surface plasmon resonance (LSPR) amplified signal from tested plasmonic NPs shows that CTAB-AuNPs was far superior in amplifying the fluorescence signal of the nanoprobe. A detection limit of 4.6 nM (1.56 ng.mL −1), rapid response time (~2 min) and excellent selectivity against other drugs, substances and cocaine metabolites was achieved. The strep-B-QD-GO-CTAB-AuNP aptamer-based fluorescent nanoprobe was successfully applied for the determination of cocaine in seized adulterated cocaine samples.</p

Fluorescence Sensing Using DNA Aptamers in Cancer Research and Clinical Diagnostics

Among the various advantages of aptamers over antibodies, remarkable is their ability to tolerate a large number of chemical modifications within their backbone or at the termini without losing significant activity. Indeed, aptamers can be easily equipped with a wide variety of reporter groups or coupled to different carriers, nanoparticles, or other biomolecules, thus producing valuable molecular recognition tools effective for diagnostic and therapeutic purposes. This review reports an updated overview on fluorescent DNA aptamers, designed to recognize significant cancer biomarkers both in soluble or membrane-bound form. In many examples, the aptamer secondary structure switches induced by target recognition are suitably translated in a detectable fluorescent signal using either fluorescently-labelled or label-free aptamers. The fluorescence emission changes, producing an enhancement ("signal-on") or a quenching ("signal-off") effect, directly reflect the extent of the binding, thereby allowing for quantitative determination of the target in bioanalytical assays. Furthermore, several aptamers conjugated to fluorescent probes proved to be effective for applications in tumour diagnosis and intraoperative surgery, producing tumour-type specific, non-invasive in vivo imaging tools for cancer pre- and post-treatment assessment

Tb3+-Cleavage Assays Reveal Specific Mg2+ Binding Sites Necessary to Pre-fold the btuB Riboswitch for AdoCbl Binding

Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg2+ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg2+ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb3+ being an established Mg2+ mimic. Interestingly many of the Mn+ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B12-binding riboswitches. Comparison with the published crystal structure of the coenzyme B12 riboswitch of S. thermophilum aided in identifying a common set of Mn+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mn+ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mn+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb3+ mediated footprints of the riboswitch's structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement

Aptamers in oncology: a diagnostic perspective

Nucleic acid sequences can produce a wide variety of three-dimensional conformations. Some of these structural forms are able to interact with proteins and small molecules with high affinity and specificity. These sequences, comprising either double or single stranded oligonucleotides, are called 'aptamers' based on the Greek word aptus, which means 'to fit'. Using an efficient selection process, randomised oligonucleotide libraries can be rapidly screened for aptamers with the appropriate binding characteristics. This technology has spawned the development of a new class of oligonucleotide therapeutic products. However, while interest among pharmaceutical companies continues to grow with some candidates already in clinical trials and one in the market, there appears to be some reluctance to fully explore the diagnostic potential of this technology. This article will review aptamer developments in diagnostics, compare them with other oligonucleotide therapeutics and highlight both potentials and pitfalls of technological development in this area

Aptamer based electrochemical sensors for emerging environmental pollutants

International audienceEnvironmental contaminants monitoring is one of the key issues in understanding and managing hazards to human health and ecosystems. In this context, aptamer based electrochemical sensors have achieved intense significance because of their capability to resolve a potentially large number of problems and challenges in environmental contamination. An aptasensor is a compact analytical device incorporating an aptamer (oligonulceotide) as the sensing element either integrated within or intimately associated with a physiochemical transducer surface. Nucleic acid is well known for the function of carrying and passing genetic information, however, it has found a key role in analytical monitoring during recent years. Aptamer based sensors represent a novelty in environmental analytical science and there are great expectations for their promising performance as alternative to conventional analytical tools. This review paper focuses on the recent advances in the development of aptamer based electrochemical sensors for environmental applications with special emphasis on emerging pollutants

G-quadruplex DNA aptamers and their ligands: Structure, function and application

Highly specific and tight-binding nucleic acid aptamers have been selected against a variety of molecular targets for over 20 years. A significant proportion of these oligonucleotides display G-quadruplex structures, particularly for DNA aptamers, that enable molecular recognition of their ligands. G-quadruplex structures couple a common scaffold to varying loop motifs that act in target recognition. Here, we review DNA G-quadruplex aptamers and their ligands from a structural and functional perspective. We compare the diversity of DNA G-quadruplex aptamers selected against multiple ligand targets, and consider structure with a particular focus on dissecting the thrombin binding aptamer - thrombin interaction. Therapeutic and analytical applications of DNA G-quadruplex aptamers are also discussed. Understanding DNA G-quadruplex aptamers carries implications not only for therapeutics and diagnostics, but also in the natural biochemistry of guanine-rich nucleic acids. © 2012 Bentham Science Publishers.postprin

eEF1A mediates the nuclear export of SNAG-containing proteins via the exportin5-aminoacyl-tRNA complex

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License.Exportin5 mediates the nuclear export of double-stranded RNAs, including pre-microRNAs, adenoviral RNAs, and tRNAs. When tRNAs are aminoacylated, the Exportin5-aminoacyl (aa)-tRNA complex recruits and coexports the translation elongation factor eEF1A. Here, we show that eEF1A binds to Snail transcription factors when bound to their main target, the E-cadherin promoter, facilitating their export to the cytoplasm in association with the aa-tRNA-Exportin5 complex. Snail binds to eEF1A through the SNAG domain, a protein nuclear export signal present in several transcription factor families, and this binding is regulated by phosphorylation. Thus, we describe a nuclear role for eEF1A and provide a mechanism for protein nuclear export that attenuates the activity of SNAG-containing transcription factors. © 2013 The Authors.This work was supported by grants from the Spanish Ministry of Science and Innovation (SAF2010-21143 to A.C.; BFU2008-01042 and CONSOLIDER-INGENIO 2010 CSD2007-00023 to M.A.N.; and CDS2007-00017 to A.C. and M.A.N.’s lab) and the Generalitat Valenciana (Prometeo 2008/049 and PROMETEOII/2013/002 to M.A.N.).Peer Reviewe

Identification and characterization of aptameric inhibitors of human neutrophil elastase

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation and tissue remodelling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used systematic evolution of ligands by exponential enrichment (SELEX) to develop single-stranded DNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency, and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models