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.

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.

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.

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.

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.

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.

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.

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.

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.

Stabilized Interleukin-6 receptor binding RNA aptamers.

Interleukin-6 (IL-6) is a multifunctional cytokine that is involved in the progression of various inflammatory diseases, such as rheumatoid arthritis and certain cancers; for example, multiple myeloma or hepatocellular carcinoma. To interfere with IL-6-dependent diseases, targeting IL-6 receptor (IL-6R)-presenting tumor cells using aptamers might be a valuable strategy to broaden established IL-6- or IL-6R-directed treatment regimens. Recently, we reported on the in vitro selection of RNA aptamers binding to the human IL-6 receptor (IL-6R) with nanomolar affinity. One aptamer, namely AIR-3A, was 19 nt in size and able to deliver bulky cargos into IL-6R-presenting cells. As AIR-3A is a natural RNA molecule, its use for in vivo applications might be limited due to its susceptibility to ubiquitous ribonucleases. Aiming at more robust RNA aptamers targeting IL-6R, we now report on the generation of stabilized RNA aptamers for potential in vivo applications. The new 2'-F-modified RNA aptamers bind to IL-6R via its extracellular portion with low nanomolar affinity comparable to the previously identified unmodified counterpart. Aptamers do not interfere with the IL-6 receptor complex formation. The work described here represents one further step to potentially apply stabilized IL-6R-binding RNA aptamers in IL-6R-connected diseases, like multiple myeloma and hepatocellular carcinoma.

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.

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.

N-glycosylations of human α1,3-fucosyltransferase IX are required for full enzyme activity.

Human α1,3-fucosyltransferase IX catalyzes the transfer of l-fucose from guanosine diphosphate-ß-L-fucose to N-acetyllactosamine, generating a Lewis X epitope, and is thereby involved in the synthesis of fucosylated cell surface glycoconjugates. It contains three putative N-glycosylation sites (Asn62, Asn101 and Asn153). The current study considers the functional role of these potential N-glycosylations within the enzyme. We produced truncated variants of human fucosyltransferase IX containing the soluble extracellular catalytic domain. To analyze the relevance of each N-glycosylation site, several genomic mutant DNAs encoding a glutamine (Gln/Q) instead of the asparagine residue were created prosperously using site-directed mutagenesis and subsequently expressed in Spodoptera frugiperda cells applying a baculovirus expression system. After production and purification of these variants of human FucT IX, the wild-type (wt) enzyme and the variants were characterized regarding their activity and kinetic properties. The variants showed lower activity than the wt FucT, whereas the individual N-glycosylation sites had different effects on the enzyme activity and kinetic parameters. While the single variant N62Q still showed ∼60% of wt activity and N101Q retained ∼30% activity, replacement of Asn153 by glutamine led to an almost complete loss of enzymatic activity. The same could be observed for variants missing two or more putative N-glycosylation sites, which indicated the importance of N-glycosylation for enzyme stability and activity.

Fluorescently labeled substrates for monitoring α1,3-fucosyltransferase IX activity.

Fucosylation is often the final process in glycan biosynthesis. The resulting glycans are involved in a variety of biological processes, such as cell adhesion, inflammation, or tumor metastasis. Fucosyltransferases catalyze the transfer of fucose residues from the activated donor molecule GDP-ß-L-fucose to various acceptor molecules. However, detailed information about the reaction processes is still lacking for most fucosyltransferases. In this work we have monitored α1,3-fucosyltransferase activity. For both donor and acceptor substrates, the introduction of a fluorescent ATTO dye was the last step in the synthesis. The subsequent conversion of these substrates into fluorescently labeled products by α1,3-fucosyltransferases was examined by high-performance thin-layer chromatography coupled with mass spectrometry as well as dual-color fluorescence cross-correlation spectroscopy, which revealed that both fluorescently labeled donor GDP-ß-L-fucose-ATTO 550 and acceptor N-acetyllactosamine-ATTO 647N were accepted by recombinant human fucosyltransferase IX and Helicobacter pylori α1,3-fucosyltransferase, respectively. Analysis by fluorescence cross-correlation spectroscopy allowed a quick and versatile estimation of the progress of the enzymatic reaction and therefore this method can be used as an alternative method for investigating fucosyltransferase reactions.

d(GGGT) 4 and r(GGGU) 4 are both HIV-1 inhibitors and interleukin-6 receptor aptamers.

Aptamers are oligonucleotides that bind targets with high specificity and affinity. They have become important tools for biosensing, target detection, drug delivery and therapy. We selected the quadruplex-forming 16-mer DNA aptamer AID-1 [d(GGGT) 4] with affinity for the interleukin-6 receptor (IL-6R) and identified single nucleotide variants that showed no significant loss of binding ability. The RNA counterpart of AID-1 [r(GGGU) 4] also bound IL-6R as quadruplex structure. AID-1 is identical to the well-known HIV inhibitor T30923, which inhibits both HIV infection and HIV-1 integrase. We also demonstrated that IL-6R specific RNA aptamers not only bind HIV-1 integrase and inhibit its 3' processing activity in vitro, but also are capable of preventing HIV de novo infection with the same efficacy as the established inhibitor T30175. All these aptamer target interactions are highly dependent on formation of quadruplex structure.

A fluorescence correlation spectroscopy-based enzyme assay for human Dicer.

Here, we present an in vitro assay based on fluorescence correlation spectroscopy (FCS), which allows investigation of the kinetic behaviour of human Dicer. The assay is based on the different mobilities of substrate and product. The change of substrate mobility was independent of the choice of the fluorescence label, allowing exclusion of non-specific photophysical artefacts. Dicer and RNase III cleavage led to different product diffusion times. Single-stranded RNA did not change its mobility after cleavage by both double-strand-specific RNases. In agreement with the literature, the RNase activity of Dicer could be inhibited by substituting Ca²âº for Mg²âº. In a defined system of two diffusion species of similar label and mobility differences, such as substrate and product, the linearity of the assay could be proven. An FCS-based enzyme assay is proposed, which allows monitoring of Dicer activity with high specificity in vitro.

The small RNA RybA regulates key-genes in the biosynthesis of aromatic amino acids under peroxide stress in E. coli.

In bacteria, adaptive response to external stimuli is often regulated by small RNAs (sRNAs). In Escherichia coli, the organism in which sRNAs have been best characterized so far, no function could be attributed to 40 out of 79 sRNAs. Here we decipher the function of RybA, one of these orphan sRNAs. RybA was discovered in 2001 by Wassarman et al. using comparative genomics. This sRNA is conserved between E. coli, Salmonella typhimurium and Klebsiella pneumoniae. We determined the expression pattern of RybA under different growth conditions and identified its exact 5' and 3' ends. Using microarray and Northern analysis we show that, under peroxide stress, the absence of RybA leads to an upregulation of key genes of the TyrR regulon involved in the metabolism of aromatic compounds including the aromatic amino acids. Although containing an open reading frame, which might have an independent function, RybA does not require translation for this activity and therefore acts at the RNA level. Furthermore we demonstrate that regulation requires the transcription regulator TyrR. The mechanism of activation of TyrR, probably the primary target of RybA, remains to be elucidated. The downregulation of aromatic amino acid biosynthesis might regulate the cellular concentration of chorismate and its availability for other downstream products like ubiquinone or enterobactin. While ubiquinone participates in the defense against oxidative stress in the cytoplasmic membrane, enterobactin is involved in iron import and is therefore detrimental under oxidative stress.

Interleukin-6 receptor specific RNA aptamers for cargo delivery into target cells.

Aptamers represent an emerging strategy to deliver cargo molecules, including dyes, drugs, proteins or even genes, into specific target cells. Upon binding to specific cell surface receptors aptamers can be internalized, for example by macropinocytosis or receptor mediated endocytosis. Here we report the in vitro selection and characterization of RNA aptamers with high affinity (Kd = 20 nM) and specificity for the human IL-6 receptor (IL-6R). Importantly, these aptamers trigger uptake without compromising the interaction of IL-6R with its natural ligands the cytokine IL-6 and glycoprotein 130 (gp130). We further optimized the aptamers to obtain a shortened, only 19-nt RNA oligonucleotide retaining all necessary characteristics for high affinity and selective recognition of IL-6R on cell surfaces. Upon incubation with IL-6R presenting cells this aptamer was rapidly internalized. Importantly, we could use our aptamer, to deliver bulky cargos, exemplified by fluorescently labeled streptavidin, into IL-6R presenting cells, thereby setting the stage for an aptamer-mediated escort of drug molecules to diseased cell populations or tissues.

RNA dimerization monitored by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) provides a versatile tool to investigate molecular interaction under native conditions, approximating infinite dilution. One precondition for its application is a sufficient difference between the molecular weights of the fluorescence-labelled unbound and bound ligand. In previous studies, an 8-fold difference in molecular weights or correspondingly a 1.6-fold difference in diffusion coefficients was required to accurately distinguish between two diffusion species by FCS. In the presented work, the hybridization of two complementary equally sized RNA single strands was investigated at an excellent signal-to-noise ratio enabled by the highly photostable fluorophore Atto647N. The fractions of ssRNA and dsRNA were quantified by applying multicomponent model analysis of single autocorrelation functions and globally fitting several autocorrelation functions. By introducing a priori knowledge into the fitting procedure, 1.3- to 1.4-fold differences in diffusion coefficients of single- and double-stranded RNA of 26, 41, and 54 nucleotides could be accurately resolved. Global fits of autocorrelation functions of all titration steps enabled a highly accurate quantification of diffusion species fractions and mobilities. At a high signal-to-noise ratio, the median of individually fitted autocorrelation functions allowed a robust representation of heterogeneous data. These findings point out the possibility of studying molecular interaction of equally sized molecules based on their diffusional behavior, which significantly broadens the application spectrum of FCS.

Fluorophore binding aptamers as a tool for RNA visualization.

Fluorescence correlation spectroscopy (FCS) is suitable for the detection of fluorescent molecules in living cells. For the visualization of mRNA, we genetically fused a fluorophore-specific RNA aptamer to the coding mRNA of the green fluorescent protein, as well as to noncoding sequences. Using these constructs, we showed that the aptamer portion of the mRNA still binds the fluorophore in the nanomolar range as determined via FCS. Furthermore, the binding took place in the context of total RNA extract. A tandem construct of the RNA aptamer even exhibited a lower K(d) than the monomer. This FCS-based method establishes a tool for minimal invasive detection of RNA at the single molecule level in individual living cells.