Aptamer selection against cell extracts containing the zoonotic obligate intracellular bacterium, Anaplasma phagocytophilum.

A. phagocytophilum is a zoonotic and tick-borne bacterium, threatening human and animal health. Many questions persist concerning the variability of strains and the mechanisms governing the interactions with its different hosts. These gaps can be explained by the difficulty to cultivate and study A. phagocytophilum because of its strict intracellular location and the lack of specific tools, in particular monoclonal antibodies, currently unavailable. The objective of our study was to develop DNA aptamers against A. phagocytophilum, or molecules expressed during the infection, as new study and/or capture tools. Selecting aptamers was a major challenge due to the strict intracellular location of the bacterium. To meet this challenge, we set up a customized selection protocol against an enriched suspension of A. phagocytophilum NY18 strain, cultivated in HL-60 cells. The implementation of SELEX allowed the selection of three aptamers, characterized by a high affinity for HL-60 cells infected with A. phagocytophilum NY18 strain. Interestingly, the targets of these three aptamers are most likely proteins expressed at different times of infection. The selected aptamers could contribute to increase our understanding of the interactions between A. phagocytophilum and its hosts, as well as permit the development of new diagnostic, therapeutic or drug delivery appliances.

The forkhead DNA-binding domain binds specific G2-rich RNA sequences.

Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed of dozens of paralogs in mammals. The forkhead domain (FHD) is a segment of about 100 amino acids that binds an A-rich DNA sequence. Using DNA and RNA PCR-SELEX, we show that recombinant FOXL2 proteins, either wild-type or carrying the oncogenic variant C134W, recognize similar DNA-binding sites. This suggests that the oncogenic variant does not alter the intrinsic sequence-specificity of FOXL2. Most importantly, we show that FOXL2 binds G2-rich RNA sequences whereas it virtually fails to bind similar sequences in DNA chemistry. Interestingly, a statistically significant subset of genes responding to the knock-down of FOXL2/Foxl2 harbor such G2-rich sequences and are involved in crucial signaling pathways and cellular processes. In addition, we show that FOXA1, FOXO3a and chimeric FOXL2 proteins containing the FHD of the former are also able to interact with some of the preferred FOXL2-binding sequences. Our results point to an unexpected and novel characteristic of the forkhead domain, the biological relevance of which remains to be explored.

Identification and Characterization of Aptamers Targeting Ovarian Cancer Biomarker Human Epididymis Protein 4 for the Application in Urine.

Ovarian 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.

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

Although 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.

Aptamer-Based Imaging of Polyisoprenoids in the Malaria Parasite.

Dolichols 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.

Polyamine transport system-targeted nanometric micelles assembled from epipodophyllotoxin-amphiphiles.

Micelle-forming amphiphilic drug conjugates were synthesized starting from a biologically active epipodophyllotoxin derivative which was covalently inserted in between a hydrophilic targeting spermine unit, and a hydrophobic stearyl chain. The amphiphilic drug conjugates were further assembled into the corresponding micelles and evaluated in vitro for the active targeting of tumor cells overexpressing the polyamine transport system.

Real-Time Monitoring of Exosome Enveloped-AAV Spreading by Endomicroscopy Approach: A New Tool for Gene Delivery in the Brain.

Exosomes represent a strategy for optimizing the adeno-associated virus (AAV) toward the development of novel therapeutic options for neurodegenerative disorders. However, in vivo spreading of exosomes and AAVs after intracerebral administration is poorly understood. This study provides an assessment and comparison of the spreading into the brain of exosome-enveloped AAVs (exo-AAVs) or unassociated AAVs (std-AAVs) through in vivo optical imaging techniques like probe-based confocal laser endomicroscopy (pCLE) and ex vivo fluorescence microscopy. The std-AAV serotypes (AAV6 and AAV9) encoding the GFP were enveloped in exosomes and injected into the ipsilateral hippocampus. At 3 months post-injection, pCLE detected enhanced GFP expression of both exo-AAV serotypes in contralateral hemispheres compared to std-AAVs. Although sparse GFP-positive astrocytes were observed using exo-AAVs, our results show that the enhancement of the transgene expression resulting from exo-AAVs was largely restricted to neurons and oligodendrocytes. Our results suggest (1) the possibility of combining gene therapy with an endoscopic approach to enable tracking of exo-AAV spread, and (2) exo-AAVs allow for widespread, long-term gene expression in the CNS, supporting the use of exo-AAVs as an efficient gene delivery tool.

Tumor targeted micellar nanocarriers assembled from epipodophyllotoxin-based amphiphiles.

Micelle-forming amphiphilic drug conjugates were synthesized starting from a biologically active epipodophyllotoxin derivative which was covalently inserted in between a hydrophilic PEG unit and a hydrophobic stearyl chain. The epipodophyllotoxin-containing amphiphiles were assembled into the corresponding micelles which were evaluated in vivo for their tumor targeting properties.

Aptamer-decorated polydiacetylene micelles with improved targeting of cancer cells.

In this study, a "click and hybridization" strategy was developed for the functionalization of polydiacetylene micelles with a targeting aptamer ligand. Decoration of the nanocarriers with an anti-Annexin A2 sequence efficiently triggered enhanced internalization of the functionalized micelles in the MCF-7 cell line, with a marked increase compared to control micelles.

Targeting hormone refractory prostate cancer by in vivo selected DNA libraries in an orthotopic xenograft mouse model.

The targeting of specific tissue is a major challenge for the effective use of therapeutics and agents mediating this targeting are strongly demanded. We report here on an in vivo selection technology that enables the de novo identification of pegylated DNA aptamers pursuing tissue sites harbouring a hormone refractory prostate tumour. To this end, two libraries, one of which bearing an 11 kDa polyethylene glycol (PEG) modification, were used in an orthotopic xenograft prostate tumour mouse model for the selection process. Next-generation sequencing revealed an in vivo enriched pegylated but not a naïve DNA aptamer recognising prostate cancer tissue implanted either subcutaneous or orthotopically in mice. This aptamer represents a valuable and cost-effective tool for the development of targeted therapies for prostate cancer. The described selection strategy and its analysis is not limited to prostate cancer but will be adaptable to various tissues, tumours, and metastases. This opens the path towards DNA aptamers being experimentally and clinically engaged as molecules for developing targeted therapy strategies.

Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau Proteins.

Herein, we report for the first time the isolation of DNA aptamers directed against the whole tau protein, an important Alzheimer's disease (AD) biomarker. Non-SELEX approach based on the capillary electrophoresis partitioning technique was employed to isolate a high-affinity DNA sequence pool towards the target in only three rounds and one working day. High-throughput sequencing was next performed and the recognition ability of five selected aptamers was preliminary evaluated by surface plasmon resonance using the protein target immobilized on the chip. Finally, the analytical potential of the most affine aptamer was demonstrated through the design of a homogeneous-phase fluorescence anisotropy assay. This DNA aptamer was found to be able to recognize not only the whole τ-441 but also the τ-381, τ-352, τ-383 isoforms. The sensing platform allowed the determination of these four targets with a detection limit of 28 nM, 3.2 nM, 6.3 nM and 22 nM, respectively.

Subtle changes in network composition impact the biodistribution and tumor accumulation of nanogels.

We studied the effect of subtle changes in side-chain chemistry and labelling with near infrared fluorophores of nanogels (NGs) prepared from thiolated poly(glycidol) on in vivo biodistribution in mice bearing human breast tumor xenografts. The stability and amphiphilic character of the side chain as well as labelling clearly influenced tumor targeting and overall biodistribution.

Application of aptamers for in vivo molecular imaging and theranostics.

Nucleic acid aptamers are small three-dimensional structures of oligonucleotides selected to bind to a target of interest with high affinity and specificity. In vitro, aptamers already compete with antibodies to serve as imaging probes, e.g. for microscopy or flow cytometry. However, they are also increasingly used for in vivo molecular imaging. Accordingly, aptamers have been evaluated over the last twenty years in almost every imaging modality, including single photon emission computed tomography, positron emission tomography, magnetic resonance imaging, fluorescence imaging, echography, and x-ray computed tomography. This review focuses on the studies that were conducted in vivo with aptamer-based imaging probes. It also presents how aptamers have been recently used to develop new types of probes for multimodal imaging and theranostic applications.

Time-lapse imaging of molecular evolution by high-throughput sequencing.

High-throughput sequencing of in vitro selection could artificially provide large quantities of relic sequences from known times of molecular evolution. Here, we demonstrate how it can be used to reconstruct an empirical genealogical evolutionary (EGE) tree of an aptamer family. In contrast to classical phylogenetic trees, this tree-diagram represents proliferation and extinction of sequences within a population during rounds of selection. Such information, which corresponds to their evolutionary fitness, is used to infer which sequences may have been mutated through the selection process that led to the appearance and spreading of new sequences. This approach was validated by the re-analysis of an in vitro selection that had previously identified an aptamer against Annexin A2. It revealed that this aptamer might be the descendant of a sequence that was more highly amplified in early rounds. It also succeeded in predicting improved variants of this aptamer and providing a means to understand the influence of selection pressure on evolution. This is the first demonstration that HTS can provide time-lapse imaging of the evolutionary pathway that is taken by a macromolecule during in vitro selection to evolve by successive mutations through better fitness.

Biotin-functionalized targeted polydiacetylene micelles.

Polydiacetylene micelles were functionalized with controlled amounts of biotin using bioorthogonal click chemistry. The biotinylated micelles were evaluated in the selective targeting of the MCF-7 cancerous cell line and were shown to be readily internalized. The efficiency of the cellular uptake was correlated to the density of grafted biotin.

Nucleic acid aptamers for neurodegenerative diseases.

The 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.

Selection of Aptamers Against Whole Living Cells: From Cell-SELEX to Identification of Biomarkers.

Aptamer selection protocols, named cell-SELEX, have been developed to target trans-membrane proteins using whole living cells as target. This technique presents several advantages. (1) It does not necessitate the use of purified proteins. (2) Aptamers are selected against membrane proteins in their native conformation. (3) Cell-SELEX can be performed to identify aptamers against biomarkers differentially expressed between different cell lines without prior knowledge of the targets. (4) Aptamers identified by cell-SELEX can be further used to purify their targets and to identify new biomarkers. Here, we provide a protocol of cell-SELEX including the preparation of an oligonucleotide library, next-generation sequencing and radioactive binding assays. Furthermore, we also provide a protocol to purify and identify the target of these aptamers. These protocols could be useful for the discovery of lead therapeutic compounds and diagnostic cell-surface biomarkers.

DNA aptamer affinity ligands for highly selective purification of human plasma-related proteins from multiple sources.

Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general.

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/).

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.