Neutralizing Aptamers from Whole-Cell SELEX Inhibit the RET Receptor Tyrosine Kinase

Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance

Aptamers in Bordeaux, 24−25 June 2016

The symposium covered the many different aspects of the selection and the characterization of aptamers as well as their application in analytical, diagnostic and therapeutic areas. Natural and artificial riboswitches were discussed. Recent advances for the design of mutated polymerases and of chemically modified nucleic acid bases that provide aptamers with new properties were presented. The power of aptamer platforms for multiplex analysis of biomarkers of major human diseases was described. The potential of aptamers for the treatment of cancer or cardiovascular diseases was also presented. Brief summaries of the lectures presented during the symposium are given in this report. A second edition of “Aptamers in Bordeaux” will take place on September 2017 (http://www.aptamers-in-bordeaux.com/)

Aptamers for Molecular Imaging

International audienceNucleic acid aptamers have been selected against a wide variety of targets and are increasingly used in biotechnological applications where they are often compared to “chemical antibodies” Because of their high specificity and affinity, aptamers are promising tools for molecular imaging. They have been evaluated over the last 20 years in almost every imaging modality, including single photon emission computed tomography, positron emission tomography, fluorescence imaging, magnetic resonance imaging, X-ray computed tomography, and ultrasound imaging. This chapter explains the different processes that are used to develop a molecular imaging probe with an aptamer, presenting its selection, optimization, labeling, and preclinical evaluation by molecular imaging. It also discusses potential pitfalls that may be encountered and important controls to be applied

Improvement of Aptamers by High-Throughput Sequencing of Doped-SELEX

International audienceAlthough SELEX can identify high-affinity aptamers, Doped-SELEX is often performed post-selection for the identification of better variants. Starting from a partially randomized (doped) library derived from an already identified aptamer, this method can screen rapidly several thousand substitutions in order to identify those that can improve the binding of the aptamers. It can also highlight the positions that do not tolerate substitutions, which suggest they are crucial for the interaction of the aptamer with its target. High-throughput sequencing (HTS), also named next-generation sequencing (NGS), can dramatically improve this method by studying millions of sequences. This high number of sequences ensures a statistically robust analysis of variants even for those with a low frequency in the library. It can reduce the number of selection rounds and provide a more in-depth analysis of the positions that are crucial for the aptamer affinity. In this chapter, we provide a protocol to simultaneously study and improve an aptamer using Doped-SELEX and HTS analysis, including the design of the doped library, the selection, HTS, and analysis. This protocol could be useful to improve the affinity of an aptamer and to reduce its size as well as to improve ribozyme

Nucleic acid aptamers for neurodegenerative diseases

International audienceThe increased incidence of neurodegenerative diseases represents a huge challenge for societies. These diseases are characterized by neuronal death and include several different pathologies, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease and transmissible spongiform encephalopathies. Most of these pathologies are often associated with the aggregation of misfolded proteins, such as amyloid-ß, tau, α-synuclein, huntingtin and prion proteins. However, the precise mechanisms that lead to neuronal dysfunction and death in these diseases remain poorly understood. Nucleic acid aptamers represent a new class of ligands that could be useful to better understand these diseases and develop better diagnosis and therapy. In this review, several of these aptamers are presented as well as their applications for neurodegenerative diseases

Applications of High-Throughput Sequencing for In Vitro Selection and Characterization of Aptamers

Aptamers are identified through an iterative process of evolutionary selection starting from a random pool containing billions of sequences. Simultaneously to the amplification of high-affinity candidates, the diversity in the pool is exponentially reduced after several rounds of in vitro selection. Until now, cloning and Sanger sequencing of about 100 sequences was usually used to identify the enriched candidates. However, High-Throughput Sequencing (HTS) is now extensively used to replace such low throughput sequencing approaches. Providing a deeper analysis of the library, HTS is expected to accelerate the identification of aptamers as well as to identify aptamers with higher affinity. It is also expected that it can provide important information on the binding site of the aptamers. Nevertheless, HTS requires handling a large amount of data that is only possible through the development of new in silico methods. Here, this review presents these different strategies that have been recently developed to improve the identification and characterization of aptamers using HTS

Identification et caractérisation d'aptamères ciblant le biomarqueur du cancer de l'ovaire Human Epididymis Protein 4 pour l'application dans l'urine

International audienceOvarian cancer is the deadliest gynecological cancer. With non-specific symptoms of the disease and the lack of effective diagnostic methods, late diagnosis remains the crucial hurdle of the poor prognosis. Therefore, development of novel diagnostic approaches are needed. The purpose of this study is to develop DNA-based aptamers as potential diagnostic probes to detect ovarian cancer biomarker Human epididymis protein 4 (HE4) in urine. HE4 is a protein overexpressed in ovarian cancer, but not in healthy or benign conditions. With high stability and diagnostic value for detection of ovarian cancer, urine HE4 appears as an attractive non-invasive biomarker. The high-affinity anti-HE4 DNA aptamers were selected through 10 cycles of High Fidelity Systematic Evolution of Ligands by EXponential enrichment (Hi-Fi SELEX), a method for aptamer selection based on digital droplet PCR. The anti-HE4 aptamers were identified using DNA sequencing and bioinformatics analysis. The candidate aptamer probes were characterized in urine for binding to HE4 protein using thermofluorimetry. Two anti-HE4 aptamers, AHE1 and AHE3, displayed binding to HE4 protein in urine, with a constant of dissociation in the nanomolar range, with Kd (AHE1) = 87 ± 9 nM and Kd (AHE3) aptamer of 127 ± 28 nM. Therefore, these aptamers could be promising tools for application in diagnostics and future development of urine tests or biosensors for ovarian cancer.Le cancer de l'ovaire est le cancer gynécologique le plus mortel. Avec des symptômes non spécifiques de la maladie et le manque de méthodes de diagnostic efficaces, le diagnostic tardif reste l'obstacle crucial du mauvais pronostic. Par conséquent, le développement de nouvelles approches diagnostiques est nécessaire. Le but de cette étude est de développer des aptamères à base d'ADN comme sondes diagnostiques potentielles pour détecter le biomarqueur du cancer de l'ovaire, la protéine 4 de l'épididyme humain (HE4) dans l'urine. HE4 est une protéine surexprimée dans le cancer de l'ovaire, mais pas dans des conditions saines ou bénignes. Avec une grande stabilité et une valeur diagnostique pour la détection du cancer de l'ovaire, l'HE4 urinaire apparaît comme un biomarqueur non invasif attractif. Les aptamères d'ADN anti-HE4 de haute affinité ont été sélectionnés par 10 cycles d'évolution systématique haute fidélité des ligands par enrichissement EXponentiel (Hi-Fi SELEX), une méthode de sélection des aptamères basée sur la PCR numérique en gouttelettes. Les aptamères anti-HE4 ont été identifiés par séquençage de l'ADN et analyse bioinformatique. Les sondes aptamères candidates ont été caractérisées dans l'urine pour la liaison à la protéine HE4 par thermofluorimétrie. Deux aptamères anti-HE4, AHE1 et AHE3, ont montré une liaison à la protéine HE4 dans l'urine, avec une constante de dissociation dans la gamme nanomolaire, avec Kd (AHE1) = 87 ± 9 nM et Kd (AHE3) aptamère de 127 ± 28 nM. Par conséquent, ces aptamères pourraient être des outils prometteurs pour une application dans le diagnostic et le développement futur de tests urinaires ou de biocapteurs pour le cancer de l'ovaire

Aptamer-Based Imaging of Polyisoprenoids in the Malaria Parasite

International audienceDolichols are isoprenoid end-products of the mevalonate and 2C-methyl-D-erythritol-4-phosphate pathways. The synthesis of dolichols is initiated with the addition of several molecules of isopentenyl diphosphate to farnesyl diphosphate. This reaction is catalyzed by a cis-prenyltransferase and leads to the formation of polyprenyl diphosphate. Subsequent steps involve the dephosphorylation and reduction of the α-isoprene unit by a polyprenol reductase, resulting in the generation of dolichol. The size of the dolichol varies, depending on the number of isoprene units incorporated. In eukaryotes, dolichols are synthesized as a mixture of four or more different lengths. Their biosynthesis is predicted to occur in the endoplasmic reticulum, where dolichols play an essential role in protein glycosylation. In this study, we have developed a selection of aptamers targeting dolichols and enhanced their specificity by incorporating fatty acids for negative selection. One aptamer showed high enrichment and specificity for linear polyisoprenoids containing at least one oxygen atom, such as an alcohol or aldehyde, in the α-isoprene unit. The selected aptamer proved to be a valuable tool for the subcellular localization of polyisoprenoids in the malaria parasite. To the best of our knowledge, this is the first time that polyisoprenoids have been localized within a cell using aptamer-based imaging techniques