DNA Aptamers for the Malignant Transformation Marker CD24.

Cluster of differentiation 24 (CD24) is a cell surface glycoprotein, which is largely present on hematopoietic cells and many types of solid tumor cells. CD24 is known to be involved in a wide range of downstream signaling pathways and neural development, yet the underlying mechanisms are poorly understood. Moreover, its production correlates with poor cancer prognosis, and targeting of CD24 with different antibodies has been shown to inhibit disease progression. Nucleic acid aptamers are oligonucleotides that are selected from random DNA or RNA libraries for high affinity and specific binding to a certain target. Thus, they can be used as an alternative to antibodies. To gain an insight on CD24 role and its interaction partners, we performed several SELEX (systematic evolution of ligands by exponential enrichment) experiments to select CD24-specfiic DNA aptamers. We found that the cell-SELEX approach was the most useful and that using HT-29 cell line presenting CD24 along with CD24 knockdown HT-29 cells has selected six aptamers. For the selected aptamers, we determined dissociation constants in the nanomolar range (18-709 nM) using flow cytometry. These aptamers can be applied as diagnostic tools to track cancer progression and bear a potential for therapeutic use for inhibiting signaling pathways that promote the metastatic process.

SDA and IDA - Two aptamers to inhibit cancer cell adhesion.

Aptamers which bind to proteins involved in cell-cell interactions could have significant value to directly affect cancer cell adhesion or for directed cargo delivery. Here, I discuss two aptamers: aptamer SDA which binds to E- and P-selectin, and aptamer IDA which binds to α6ß4 integrin. Both aptamers (SDA 91 nt and IDA 77 nt) bind their target proteins with dissociation constants in the 100-150 nM range and substantially inhibit special cellular adhesion, possibly a first and pivotal step in transendothelial migration during metastasis formation. The aptamers' half-lives in cell culture media are between two and six hours. IDA is internalized by integrin presenting cells within minutes thus possibly serving as vehicle for directed cargo delivery.

Charomers-Interleukin-6 Receptor Specific Aptamers for Cellular Internalization and Targeted Drug Delivery.

Interleukin-6 (IL-6) is a key player in inflammation and the main factor for the induction of acute phase protein biosynthesis. Further to its central role in many aspects of the immune system, IL-6 regulates a variety of homeostatic processes. To interfere with IL-6 dependent diseases, such as various autoimmune diseases or certain cancers like multiple myeloma or hepatocellular carcinoma associated with chronic inflammation, it might be a sensible strategy to target human IL-6 receptor (hIL-6R) presenting cells with aptamers. We therefore have selected and characterized different DNA and RNA aptamers specifically binding IL-6R. These IL-6R aptamers, however, do not interfere with the IL-6 signaling pathway but are internalized with the receptor and thus can serve as vehicles for the delivery of different cargo molecules like therapeutics. We succeeded in the construction of a chlorin e6 derivatized aptamer to be delivered for targeted photodynamic therapy (PDT). Furthermore, we were able to synthesize an aptamer intrinsically comprising the cytostatic 5-Fluoro-2'-deoxy-uridine for targeted chemotherapy. The α6ß4 integrin specific DNA aptamer IDA, also selected in our laboratory is internalized, too. All these aptamers can serve as vehicles for targeted drug delivery into cells. We call them charomers-in memory of Charon, the ferryman in Greek mythology, who ferried the deceased into the underworld.

Size dependent targeted delivery of gold nanoparticles modified with the IL-6R-specific aptamer AIR-3A to IL-6R-carrying cells.

The delivery of gold nanoparticles (AuNPs) to specific cells strongly depends on the properties e.g. the size of the particles and is of great interest for a large variety of biomedical applications. Here we investigated the size dependence of the receptor-ligand mediated AuNP delivery to cells by comparing very small "molecular" Au-clusters of only 2 nm to larger 7 nm and 36 nm AuNPs with a distinct surface plasmon resonance. Since the molecular weight in this range changes by almost three orders of magnitude, we show how the amount of gold relates to the number of delivered AuNPs. We attached small interleukin-6 receptor (IL-6R) specific aptamer molecules (AIR-3A) in different amounts to the particles and investigated the specificity of the delivery to IL-6R-carrying cells. To reduce unspecific interaction the particles were additionally covered with polyethylene glycol (PEG). Besides particle size and concentration we varied additional parameters such as aptamer surface coverage as well as incubation time and temperature. We found that in particular, small particles with diameters of less than 2 nm show an up to six times higher delivery rate for the aptamer-conjugated AuNPs compared to untargeted PEG-coated AuNPs. The specificity reduces with a decreasing aptamer/PEG ratio, and also with an increase in particle size where the unspecific uptake is much higher. In addition we also compared the delivery efficiency of this aptamer-mediated delivery system with an antibody-mediated system targeting the same receptor to validate the performance of this approach.

An endstation for resonant inelastic X-ray scattering studies of solid and liquid samples.

A novel experimental setup is presented for resonant inelastic X-ray scattering investigations of solid and liquid samples in the soft X-ray region for studying the complex electronic configuration of (bio)chemical systems. The uniqueness of the apparatus is its high flexibility combined with optimal energy resolution and energy range ratio. The apparatus enables investigation of chemical analyses, which reflects the chemical imprints. The endstation is composed of a main sample chamber, a sample holder for either solid or liquid jet delivery system, and a soft X-ray grating spectrometer for 210-1250 eV with a resolving power of ∼1000. It combines for the first time liquid jet technology with a soft X-ray spectrometer based on the variable line spacing principle. This setup was commissioned at the soft X-ray beamline P04 at PETRA III of the Deutsches Elektronen-Synchrotron in Hamburg which is currently the most brilliant storage-ring-based X-ray radiation source in the world. The first results of liquid and solid samples show that this setup allows the detection of photons across an energy range of ∼300 eV. This covers simultaneously the emission lines of life-important elements like carbon, nitrogen and oxygen in a shot-based procedure.

Immune Architecture of Colorectal Lung Metastases and Implications for Patient Survival.

BACKGROUND: Pulmonary metastases occur in 10-20% of patients with colorectal cancer and significantly influence long-term survival. In this study, the immunological architecture of colorectal lung in comparison to liver metastases and its impact on patient survival were examined. METHODS: Specimens of patients with colorectal lung and liver metastases were stained for HE, CD4, CD8, CD20, CD68 and CD45RO. Besides histomorphological evaluation, immunohistochemical stainings were analyzed for the respective cell numbers separately for tumor area, infiltrative margin and distant lung or liver stroma. These findings were correlated with clinical data and patient outcome. RESULTS: In colorectal lung (n = 69) in comparison to liver (n = 222) metastases, the immunological focus is located in the tumor region. A high CD4+ cell infiltration of this area is associated with prolonged survival of patients after resection of colorectal lung metastases [103 ± 33 (high) vs. 37 ± 6 months (low); p = 0.0246]. Patients who were treated with preoperative chemotherapy did not show differences in immune infiltrates compared to chemotherapy-naïve patients. CONCLUSION: Colorectal lung and liver metastases showed a distinct immunological architecture. A dense cell infiltration of colorectal lung metastases by CD4+ cells was related to prolonged patient survival. Preoperative chemotherapy did not influence cellular immune infiltrates.

Structure and target interaction of a G-quadruplex RNA-aptamer.

G-quadruplexes have recently moved into focus of research in nucleic acids, thereby evolving in scientific significance from exceptional secondary structure motifs to complex modulators of gene regulation. Aptamers (nucleic acid based ligands with recognition properties for a specific target) that form Gquadruplexes may have particular potential for therapeutic applications as they combine the characteristics of specific targeting and Gquadruplex mediated stability and regulation. We have investigated the structure and target interaction properties of one such aptamer: AIR-3 and its truncated form AIR-3A. These RNA aptamers are specific for human interleukin-6 receptor (hIL-6R), a key player in inflammatory diseases and cancer, and have recently been exploited for in vitro drug delivery studies. With the aim to resolve the RNA structure, global shape, RNA:protein interaction site and binding stoichiometry, we now investigated AIR-3 and AIR-3A by different methods including RNA structure probing, Small Angle X-ray scattering and microscale thermophoresis. Our findings suggest a broader spectrum of folding species than assumed so far and remarkable tolerance toward different modifications. Mass spectrometry based binding site analysis, supported by molecular modeling and docking studies propose a general Gquadruplex affinity for the target molecule hIL-6R.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity.

Selection and Characterization of an α6ß4 Integrin blocking DNA Aptamer.

The heterodimeric laminin receptor α6ß4 integrin plays a central role in the promotion of tumor cell growth, invasion, and organotropic metastasis. As an overproduction of the integrin is often linked to a poor prognosis, the inhibition of integrin α6ß4 binding to laminin is of high therapeutical interest. Here, we report on the combination of a cell-systematic evolution of ligands by exponential enrichment and a bead-based selection resulting in the first aptamer inhibiting the interaction between α6ß4 integrin and laminin-332. This Integrin α6ß4-specific DNA Aptamer (IDA) inhibits the adhesion of prostate cancer cells (PC-3) to laminin-332 with an IC50 value of 149 nmol/l. The Kd value concerning the aptamer's interaction with PC-3 cells amounts to 137 nmol/l. Further characterization showed specificity to α6 integrins and a half-life in murine blood plasma of 6 hours. Two truncated versions of the aptamer retained their binding capacity, but lost their ability to inhibit the interaction between laminin-332 and PC-3 cells. Confocal laser scanning microscope studies revealed that the aptamer was internalized into PC-3-cells. Therefore, in addition to the adhesion-blocking function of this aptamer, IDA could also be applied for the delivery of siRNA, microRNA or toxins to cancer cells presenting the integrin α6ß4.

SELEX of Cell-Specific RNA Aptamers.

This chapter focuses on the selection of RNA aptamers, which bind to specific cell surface components and thus can be internalized receptor mediated. Such aptamers discriminate between different tissues, e.g., detect malignant cells, and target them or induce apoptosis through drug internalization. However, before starting the selection process the choice of an ideal target can be challenging. To give an example for the selection of cell specific aptamers, we here used the interleukin-6 receptor (IL-6R) as a target, which is presented on hepatocytes, neutrophils, monocytes, and macrophages.

RAID3--An interleukin-6 receptor-binding aptamer with post-selective modification-resistant affinity.

Aptamers are an emerging class of highly specific targeting ligands. They can be selected in vitro for a large variety of targets, ranging from small molecules to whole cells. Most aptamers selected are nucleic acid-based, allowing chemical synthesis and easy modification. Although their properties make them interesting drug candidates for a broad spectrum of applications and an interesting alternative to antibodies or fusion proteins, they are not yet broadly used. One major drawback of aptamers is their susceptibility to abundant serum nucleases, resulting in their fast degradation in biological fluids. Using modified nucleic acids has become a common strategy to overcome these disadvantages, greatly increasing their half-life under cell culture conditions or even in vivo. Whereas pre-selective modifications of the initial library for aptamer selection are relatively easy to obtain, post-selective modifications of already selected aptamers are still generally very labor-intensive and often compromise the aptamers ability to bind its target molecule. Here we report the selection, characterization and post-selective modification of a 34 nucleotide (nt) RNA aptamer for a non-dominant, novel target site (domain 3) of the interleukin-6 receptor (IL-6R). We performed structural analyses and investigated the affinity of the aptamer to the membrane-bound and soluble forms (sIL-6R) of the IL-6R. Further, we performed structural analyses of the aptamer in solution using small-angle X-ray scattering and determined its overall shape and oligomeric state. Post-selective exchange of all pyrimidines against their 2'-fluoro analogs increased the aptamers stability significantly without compromising its affinity for the target protein. The resulting modified aptamer could be shortened to its minimal binding motif without loss of affinity.

Food Sensing: Aptamer-Based Trapping of Bacillus cereus Spores with Specific Detection via Real Time PCR in Milk.

Aerobic spores pose serious problems for both food product manufacturers and consumers. Milk is particularly at risk and thus an important issue of preventive consumer protection and quality assurance. The spore-former Bacillus cereus is a food poisoning Gram-positive pathogen which mainly produces two different types of toxins, the diarrhea inducing and the emetic toxins. Reliable and rapid analytical assays for the detection of B. cereus spores are required, which could be achieved by combining in vitro generated aptamers with highly specific molecular biological techniques. For the development of routine bioanalytical approaches, already existing aptamers with high affinity to B. cereus spores have been characterized by surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in terms of their dissociation constants and selectivity. Dissociation constants in the low nanomolar range (from 5.2 to 52.4 nM) were determined. Subsequently, the characterized aptamers were utilized for the establishment and validation of an aptamer-based trapping technique in both milk simulating buffer and milk with fat contents between 0.3 and 3.5%. Thereby, enrichment factors of up to 6-fold could be achieved. It could be observed that trapping protocol and characterized aptamers were fully adaptable to the application in milk. Due to the fact that aptamer selectivity is limited, a highly specific real time PCR assay was utilized following trapping to gain a higher degree of selectivity.

Tagging Glycoproteins with Fluorescently Labeled GDP-Fucoses by Using α1,3-Fucosyltransferases.

Fucose-containing glycans mediate a variety of biological processes, but there is little information on reaction processes and mechanisms mediated by fucosyltransferases. We recently reported on fluorescently labeled GDP-ß-L-fucose-ATTO 550, which enabled monitoring of α1,3-fucosyltransferase activity. Here we present an extension to the previously described results, based on the synthesis of a fluorescein-isothiocyanate (FITC)-labeled and two carboxyfluorescein-labeled (FAM-labeled) NDP-ß-L-fucose derivatives, and applied all four compounds in labeling of different glycoproteins with the aid of four different fucosyltransferases. The labeling processes were analyzed by in-gel fluorescence and fluorescence polarization measurements. Comparison with the ATTO-labeled sugar revealed that the FITC-labeled fucose was the best of these substrates, and that the bacterial enzyme HP-FucT tolerated the fluorescent substrates better than human fucosyltransferases.

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme.

Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys(13)) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes.

Synthesis and analysis of potential α1,3-fucosyltransferase inhibitors.

Fucosyltransferases catalyze the transfer of l-fucose from an activated GDP-ß-l-fucose to various acceptor molecules such as N-acetyllactosamine. Frequently fucosylation is the final step within the glycosylation machinery, and the resulting glycans are involved in various cellular processes such as cell-cell recognition, adhesion and inflammation or tumor metastasis. The selective blocking of these interactions would thus be a potential promising therapeutic strategy. The syntheses and analyses of various potential α1,3-fucosyltransferase inhibitors derived from GDP-ß-l-fucose containing a triazole linker unit is summarized and the observed inhibitory effect was compared with that of small molecules such as GDP or fucose. To examine their specificity and selectivity, all inhibitors were tested with human α1,3-fucosyltransferase IX and Helicobacter pylori α1,3-fucosyltransferase, which is to date the only α1,3-fucosyltransferase with a known high resolution structure. Specific inhibitors which inhibit either H. pylori α1,3-fucosyltransferase or human fucosyltransferase IX with Ki values in the micromolar range were identified. In that regard, acetylated GDP-galactose derivative Ac-3 turned out to inhibit H. pylori α1,3-fucosyltransferase but not human fucosyltransferase IX, whereas GDP-6-amino-ß-l-fucose 17 showed an appreciably better inhibitory effect on fucosyltransferase IX activity than on that of H. pylori fucosyltransferase.

Aptamers as drug delivery vehicles.

The benefits of directed and selective therapy for systemic treatment are reasons for increased interest in exploiting aptamers for cell-specific drug delivery. Nucleic acid based pharmaceuticals represent an interesting and novel tool to counter human diseases. Combining inhibitory potential and cargo transfer upon internalization, nanocarriers as well as various therapeutics including siRNAs, chemotherapeutics, photosensitizers, or proteins can be imported via these synthetic nucleic acids. However, widespread clinical application is still hampered by obstacles that must be overcome. In this review, we give an overview of applications and recent advances in aptamer-mediated drug delivery. We also introduce prominent selection methods as well as useful approaches in choice of drug and conjugation method. We discuss the challenges that need to be considered and present strategies that have been applied to achieve intracellular delivery of effectors transported by readily internalized aptamers.

An aptamer intrinsically comprising 5-fluoro-2'-deoxyuridine for targeted chemotherapy.

An aptamer specifically binding the interleukin-6 receptor and intrinsically comprising multiple units of the nucleoside analogue 5-fluoro-2'-deoxyuridine can exert a cytostatic effect direcly on certain cells presenting the receptor. Thus the modified aptamer fulfils the requirements for active drug targeting in an unprecedented manner. It can easily be synthesized in a single enzymatic step and it binds to a cell surface receptor that is conveyed into the lysosome. Upon degradation of the aptamer by intracellular nucleases the active drug is released within the targeted cells exclusively. In this way the aptamer acts as a prodrug meeting two major prerequisites of a drug delivery system: specific cell targeting and the controlled release of the drug triggered by an endogenous stimulus.

Postpolymerization modification using less cytotoxic activated ester polymers for the synthesis of biological active polymers.

Activated ester polymers, pioneered by Ferruti and Ringsdorf in the 1970s, are attractive polymeric materials because they can easily be converted into functional polymers by reacting with amine nucleophiles. In the present study, methyl salicylate acrylate, salicyl acrylate, and tert-butyl salicylate acrylate monomers were polymerized yielding three novel reactive precursors suitable for the postpolymerization modification with primary and secondary amines. The reactivities of poly(pentafluorophenyl acrylate), poly(methyl salicylate acrylic ester), and poly(salicyl acrylate) toward amines were compared by kinetic studies and revealed the practical applicability of salicylic acid based derivatives for efficient postpolymerization modifications. In addition, in vitro cytotoxicity of water-soluble leaving groups, pentafluorophenol and salicylic acid, as well as water-soluble polymers containing the respective activated ester groups were investigated using HeLa cells. In short, compared to the frequently used poly(pentafluorophenyl acrylate), poly(salicyl acrylate) activated ester feature a lower reactivity, but exhibit less cytotoxicity. In this respect, poly(salicyl acrylate) as reactive precursor polymers may become alternative routes for the synthesis of functional polyacrylamides when it comes to advanced applications in vivo.

SDA, a DNA aptamer inhibiting E- and P-selectin mediated adhesion of cancer and leukemia cells, the first and pivotal step in transendothelial migration during metastasis formation.

Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation in vivo. We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K(d) value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.

Chlorin e6 Conjugated Interleukin-6 Receptor Aptamers Selectively Kill Target Cells Upon Irradiation.

Photodynamic therapy (PDT) uses the therapeutic properties of light in combination with certain chemicals, called photosensitizers, to successfully treat brain, breast, prostate, and skin cancers. To improve PDT, current research focuses on the development of photosensitizers to specifically target cancer cells. In the past few years, aptamers have been developed to directly deliver cargo molecules into target cells. We conjugated the photosensitizer chlorin e6 (ce6) with a human interleukin-6 receptor (IL-6R) binding RNA aptamer, AIR-3A yielding AIR-3A-ce6 for application in high efficient PDT. AIR-3A-ce6 was rapidly and specifically internalized by IL-6R presenting (IL-6R(+)) cells. Upon light irradiation, targeted cells were selectively killed, while free ce6 did not show any toxic effect. Cells lacking the IL-6R were also not affected by AIR-3A-ce6. With this approach, we improved the target specificity of ce6-mediated PDT. In the future, other tumor-specific aptamers might be used to selectively localize photosensitizers into cells of interest and improve the efficacy and specificity of PDT in cancer and other diseases.Molecular Therapy-Nucleic Acids (2014) 3, e143; doi:10.1038/mtna.2013.70; published online 21 January 2014.