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Concept: Aptamer

148

DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology.

Concepts: DNA, Malaria, Plasmodium falciparum, Plasmodium, Plasmodium vivax, Nanotechnology, DNA nanotechnology, Aptamer

144

The selective delivery of drugs in a cell- or tissue-specific manner represents the main challenge for medical research; in order to reduce the occurrence of unwanted off-target effects. In this regard, nucleic acid aptamers have emerged as an attractive class of carrier molecules due to their ability to bind with high affinity to specific ligands; their high chemical flexibility; as well as tissue penetration capability. To date, different aptamer-drug systems and aptamer-nanoparticles systems, in which nanoparticles function together with aptamers for the targeted delivery, have been successfully developed for a wide range of therapeutics, including toxins; peptides; chemotherapeutics and oligonucleotides. Therefore, aptamer-mediated drug delivery represents a powerful tool for the safe and effective treatment of different human pathologies, including cancer; neurological diseases; immunological diseases and so on. In this review, we will summarize recent progress in the field of aptamer-mediated drug delivery and we will discuss the advantages, the achieved objectives and the challenges to be still addressed in the near future, in order to improve the effectiveness of therapies.

Concepts: Medicine, Cancer, Effect, Cure, Effectiveness, Immunology, Neurology, Aptamer

143

Nucleic acid aptamers generated through an in vitro selection are currently extensively applied as very valuable biomolecular tools thanks to their prominent advantages. Diversity of spatial structures, ease of production through chemical synthesis and a large variety of chemical modifications make aptamers convenient building blocks for the generation of multifunctional constructs. An opportunity to combine different aptamer functionalities with other molecules of interest such as reporter groups, nanoparticles, chemotherapeutic agents, siRNA or antisense oligonucleotides provides a widest range of applications of multivalent aptamers. The present review summarizes approaches to the design of multivalent aptamers, various examples of multifunctional constructs and the prospects of employing them as components of biosensors, probes for affinity capture, tools for cell research and potential therapeutic candidates.

Concepts: DNA, Time, RNA, Chemical reaction, Molecule, Chemistry, Aptamer, Oligonucleotide

28

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=3-5 μM), and relatively weak affinity (Kd=27-30 μM) 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.

Concepts: DNA, Molecular biology, Plant, Aptamer, Cytokinin, Zeatin, Coconut milk, Cytokinins

28

A novel SERS-based sandwich immunoassay using DNA aptamers, silica-encapsulated hollow gold nanospheres (SEHGNs) and a gold-patterned microarray was developed for sensitive detection of VEGF (vascular endothelial growth factor) angiogenesis protein markers. Here, a DNA aptamer conjugated to SEHGN was used as a highly reproducible SERS-encoding nanoprobe, and a hybrid microarray including hydrophilic gold wells and other hydrophobic areas was used as a SERS substrate. Target specific DNA aptamers that fold into a G-quadruplex structure were used as a target recognition unit instead of VEGF antibodies. The detection sensitivity was increased by 2 or 3 orders of magnitude over the conventional ELISA method. In particular, the dynamic concentration range was 3 or 4 orders of magnitude greater than that of conventional ELISA. The results demonstrate that this sensing strategy using DNA aptamers is a powerful platform for the design of novel immune-sensors with high performance. In particular, SERS-based detection using SEHGNs provides great promise for highly sensitive biomarker sensing with unprecedented advantages.

Concepts: Antibody, DNA, Protein, Angiogenesis, Vascular endothelial growth factor, ELISA, Immunoassay, Aptamer

27

Aptamers are single-stranded DNA or RNA molecules with a defined tertiary structure for molecular recognition. Numerous RNA aptamers with excellent binding affinity and specificity have been reported; they constitute an attractive reservoir of molecular recognition elements for biosensor development. However, RNA is relatively unstable owing to spontaneous hydrolysis and nuclease degradation. Thus, RNA aptamer-based biosensors are prone to producing false-positive signals. Here, we present an RNA aptamer biosensor design strategy that utilises an internal control to distinguish target binding from false-positive signals. The sequence of a chosen RNA aptamer is expanded so that it can form three consecutive short RNA-DNA duplexes with 1) a quencher-labelled DNA strand (Q1 DNA), 2) a dual-fluorophore-labelled DNA strand (F1 DNAF2 ) and 3) another quencher-labelled DNA strand (Q2 DNA). The addition of a target releases Q2 DNA from the duplex assembly, and produces the expected positive signal from F2 . However, the authenticity of target recognition is validated only if no signal is generated from F1 . We have successfully engineered two fluorescent reporters by using an RNA aptamer that binds thrombin and one that binds theophylline. Both reporters show the expected binding affinity and specificity, and are capable of reporting system malfunction when treated with nucleases and chemical denaturants. This strategy provides a simple and reliable way to ensure high-quality detection when RNA aptamers are employed as molecular-recognition elements.

Concepts: DNA, Protein, Gene, Molecular biology, RNA, Molecule, Nucleotide, Aptamer

27

In this paper, a high-affinity ssDNA aptamer binding to Salmonella typhimurium was obtained by a whole-bacterium-based Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure. After nine rounds of selection with S. typhimurium as the target, a highly enriched oligonucleotide pool was sequenced and then grouped into different families based on primary sequence homology and secondary structure similarity. Eleven sequences from different families were selected for further characterization via flow cytometry analysis. The results showed that the sequence ST2P demonstrates affinity for S. typhimurium much more strongly and specifically than other sequences tested. The estimated K¬d value of this particularly promising aptamer was 6.33 ± 0.58 nM. To demonstrate the potential use of the aptamers in the quantitative determination of S. typhimurium, a fluorescent bioassay with the aptamer ST2P was prepared. Under the optimal conditions, the correlation between the concentration of S. typhimurium and fluorescent signal was found to be linear within the range of 50-106 cfu/mL (R2 =0.9957). The limit of detection (LOD) of the developed method were found to be 25 cfu/mL. Our work demonstrates that this aptamer could potentially be used to improve the detection of S. typhimurium.

Concepts: DNA, Bioinformatics, Evolution, Flow cytometry, Sequence, Salmonella enterica, Aptamer, Limit of a sequence

27

In this article, high-affinity single stranded DNA (ssDNA) aptamer-targeting F(ab')2 fragments of saxitoxin (STX) antibodies were selected from a random ssDNA library by the SELEX strategy. After 16 rounds of repeated selection, the enriched ssDNA library was sequenced, and all of the sequences were carefully identified by indirect enzyme-linked assay and indirect competitive enzyme-linked assay (icELISA). The candidate aptamers in the above identification were selected for further characterization by icELISA and the equilibrium filtration method. We successfully obtained an aptamer that mimics STX in antibody binding and a substitute for STX in aptamer form has been developed. Further work is in progress aimed at using this aptamer substitute to replace the STX standard in an antibody-based, nontoxic detection method for field determination of STX in seafood products. KEYWORDS: saxitoxin; aptamer; F(ab')2 fragments; SELEX; paralytic shellfish poisoning.

Concepts: Antibody, DNA, Molecular biology, Population genetics, Seafood, Aptamer, Paralytic shellfish poisoning, Shellfish poisoning

27

Aptamer-conjugated nanoparticles (Apt-NPs) are increasingly being developed for biomedical purposes and especially for diagnosis and therapy. However, there is no quantitative study of the targeting functionality of such grafted aptamers compared to free aptamers. Thus, we report the first determination of binding parameters for Apt-NP/target complexes, thanks to a continuous frontal analysis in a microchip electrophoresis format (FACMCE), based on a methodology previously developed by our group. As a model system, the targeting ability of a lysozyme-binding aptamer conjugated to fluorescent maghemite nanoparticles (NPs) was evaluated, and showed evidence that the conjugation does not alter the affinity of this aptamer.

Concepts: Scientific method, Model organism, Sociology, Model, Quantitative research, Affinity electrophoresis, Aptamer

26

Several protein-targeted RNA aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. We examined the structural accommodation of an RNA aptamer that binds bacterial r-protein S8. The core of the primary binding site for S8 on helix 21 of 16S rRNA contains a pair of conserved base triples that mold the sugar-phosphate backbone to S8. The aptamer, which does not contain the conserved sequence motif, is specific for the rRNA binding site of S8. The protein-free RNA aptamer adopts a helical structure with multiple non-canonical base pairs. Surprisingly, binding of S8 leads to a dramatic change in the RNA conformation that restores the signature S8 recognition fold through a novel combination of nucleobase interactions. Nucleotides within the non-canonical core rearrange to create a G-(G-C) triple and a U-(A-U)-U quartet. Although native-like S8-RNA interactions are present in the aptamer-S8 complex, the topology of the aptamer RNA differs from that of the helix 21-S8 complex. This is the first example of an RNA aptamer that adopts substantially different secondary structures in the free and protein-bound states and highlights the remarkable plasticity of RNA secondary structure.

Concepts: DNA, Protein, Gene, RNA, Ribosomal RNA, Secondary structure, Nucleotide, Aptamer