Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection.

Two subunits of the ternary troponin complex, I and C, have cardiac muscle specific isoforms, and hence could be applied as highly-selective markers of acute coronary syndrome. We aimed at paving the way for the development of a robust cardiac troponin I-detecting sandwich assay by replacing antibodies with nuclease resistant aptamer analogues, so-called spiegelmers. To complement the previously generated spiegelmers that were specific for the N-terminus of cTnI, spiegelmers were selected for an amino acid stretch in the proximity of the C-terminal part of the protein by using a D-amino acid composed peptide. Following the selection, the oligonucleotides were screened by filter binding assay, and surface plasmon resonance analysis of the most auspicious candidates demonstrated that this approach could provide spiegelmers with subnanomolar dissociation constant. To demonstrate if the selected spiegelmers are functional and suitable for cTnI detection in a sandwich type arrangement, AlphaLisa technology was leveraged and the obtained results demonstrated that spiegelmers with different epitope selectivity are suitable for specific detection of cTnI protein even in human plasma containing samples. These results suggest that spiegelmers could be considered in the development of the next generation cTnI monitoring assays.

SDF-1/CXCR4/CXCR7 is pivotal for vascular smooth muscle cell proliferation and chronic allograft vasculopathy.

Chronic rejection remains a major obstacle in transplant medicine. Recent studies suggest a crucial role of the chemokine SDF-1 on neointima formation after injury. Here, we investigate the potential therapeutic effect of inhibiting the SDF-1/CXCR4/CXCR7 axis with an anti-SDF-1 Spiegelmer (NOX-A12) on the development of chronic allograft vasculopathy. Heterotopic heart transplants from H-2bm12 to B6 mice and aortic transplants from Balb/c to B6 were performed. Mice were treated with NOX-A12. Control animals received a nonfunctional Spiegelmer (revNOX-A12). Samples were retrieved at different time points and analysed by histology, RT-PCR and proliferation assay. Blockade of SDF-1 caused a significant decrease in neointima formation as measured by intima/media ratio (1.0 ± 0.1 vs. 1.8 ± 0.1, P < 0.001 AoTx; 0.35 ± 0.05 vs. 1.13 ± 0.27, P < 0.05 HTx). In vitro treatment of primary vascular smooth muscle cells with NOX-A12 showed a significant reduction in proliferation (0.42 ± 0.04 vs. 0.24 ± 0.03, P < 0.05). TGF-ß, TNF-α and IL-6 levels were significantly reduced under SDF-1 inhibition (3.42 ± 0.37 vs. 1.67 ± 0.33, P < 0.05; 2.18 ± 0.37 vs. 1.0 ± 0.39, P < 0.05; 2.18 ± 0.26 vs. 1.6 ± 0.1, P < 0.05). SDF-1/CXCR4/CXCR7 plays a critical role in the development of chronic allograft vasculopathy (CAV). Therefore, pharmacological inhibition of SDF-1 with NOX-A12 may represent a therapeutic option to ameliorate chronic rejection changes.

Intravitreal inhibition of complement C5a reduces choroidal neovascularization in mice.

PURPOSE: To investigate the influence of complement component C5a inhibition on laser-induced choroidal neovascularization (CNV) in mice using a C5a specific L-aptamer. METHODS: In C57BL/6 J mice CNV was induced by argon-laser, C5a-inhibitor (NOX-D20) was intravitreally injected in three concentrations: 0.3, 3.0, and 30 mg/ml. The unPEGylated derivate (NOX-D20001) was applied at 3.0 mg/ml; the vehicle (5 % glucose) was injected in controls. Vascular leakage was evaluated using fluorescence angiography, CNV area was examined immunohistochemically. Activated immune cells surrounding the CNV lesion and potential cytotoxicity were analyzed. RESULTS: Compared to controls, CNV areas were significantly reduced after NOX-D20 injection at a concentration of 0.3 and 3.0 mg/ml (p = 0.042; p = 0.016). NOX-D20001 significantly decreased CNV leakage but not the area (p = 0.007; p = 0.276). At a concentration of 30 mg/ml, NOX-D20 did not reveal significant effects on vascular leakage or CNV area (p = 0.624; p = 0.121). The amount of CD11b positive cells was significantly reduced after treatment with 0.3 and 3.0 mg/ml NOX-D20 (p = 0.027; p = 0.002). No adverse glial cell proliferation or increased apoptosis were observed at effective dosages. CONCLUSIONS: Our findings demonstrate that the targeted inhibition of complement component C5a reduces vascular leakage and neovascular area in laser-induced CNV in mice. NOX-D20 was proven to be an effective and safe agent that might be considered as a therapeutic candidate for CNV treatment. The deficiency of activated immune cells highlights promising new aspects in the pathology of choroidal neovascularization, and warrants further investigations.

A mixed mirror-image DNA/RNA aptamer inhibits glucagon and acutely improves glucose tolerance in models of type 1 and type 2 diabetes.

Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2'-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a Kd of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC50 of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.

Peripherally circulating ghrelin does not mediate alcohol-induced reward and alcohol intake in rodents.

BACKGROUND: Development of alcohol dependence, a chronic and relapsing disease, largely depends on the effects of alcohol on the brain reward systems. By elucidating the mechanisms involved in alcohol use disorder, novel treatment strategies may be developed. Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor 1A, acts as an important regulator of energy balance. Recently ghrelin and its receptor were shown to mediate alcohol reward and to control alcohol consumption in rodents. However, the role of central versus peripheral ghrelin for alcohol reward needs to be elucidated. METHODS: Given that ghrelin mainly is produced by peripheral organs, the present study was designed to investigate the role of circulating endogenous ghelin for alcohol reward and for alcohol intake in rodents. RESULTS: We showed that the Spiegelmer NOX-B11-2, which binds and neutralizes acylated ghrelin in the periphery with high affinity and thus prevents its brain access, does not attenuate the alcohol-induced locomotor activity, accumbal dopamine release and expression of conditioned place preference in mice. Moreover, NOX-B11-2 does not affect alcohol intake using the intermittent access 20% alcohol 2-bottle-choice drinking paradigm in rats, suggesting that circulating ghrelin does not regulate alcohol intake or the rewarding properties of alcohol. In the present study, we showed however, that NOX-B11-2 reduced food intake in rats supporting a role for circulating ghrelin as physiological regulators of food intake. Moreover, NOX-B11-2 did not affect the blood alcohol concentration in mice. CONCLUSIONS: Collectively, the past and present studies suggest that central, rather than peripheral, ghrelin signaling may be a potential target for pharmacological treatment of alcohol dependence.

Nonbiodegradable Spiegelmer-Driven Colorimetric Biosensor for Bisphenol A Detection.

Spiegelmers are enantiomers of natural D-oligonucleotides that bind to targets with distinct structures such as aptamers. The high susceptibility of natural D-form aptamers to nucleases greatly hinders their application in biological environments. Here, a nonbiodegradable spiegelmer-based platform for the sensitive detection of bisphenol A (BPA) was developed. Due to the symmetric molecule of BPA, the D-form aptamer can be directly converted into mirror forms via chemical synthesis. Aptamer-target interactions that involve chemically synthesized spiegelmers were characterized by biolayer interferometry, and their stabilities were tested in various biological fluids by exposure to nucleases. We demonstrate for the first time the use of a nuclease-resistant spiegelmer in a simple, label-free gold nanoparticle-based colorimetric assay to detect BPA in a highly sensitive and selective manner. The aptasensor exhibits an LOD of 0.057 ng/mL and dynamic range of 105 (100 pg/mL to 10 mg/mL). With sensing capacity and biological stability, the developed aptasensor shows great potential to utilize in in-field applications such as water quality monitoring.

Selection of DNA Aptamers for Root Exudate l-Serine Using Multiple Selection Strategies.

Agricultural biosensing can aid decisions about crop health and maintenance, because crops release root exudates that can inform about their status. l-Serine has been found to be indicative of nitrogen uptake in wheat and canola. The development of a biosensor for l-serine could allow farmers to monitor crop nutrient demands more precisely. The development of robust l-serine-binding DNA aptamers is described. Because small molecules can be challenging targets for Systematic Evolution of Ligands by EXponential enrichment (SELEX), three separate DNA libraries were used for SELEX experiments. A l-homocysteine aptamer was randomized to create a starting library for a l-serine selection (randomized SELEX). The final selection rounds of the l-homocysteine selection were also used as a starting library for l-serine (redirected SELEX). Finally, an original DNA library was used (original SELEX). All three SELEX experiments produced l-serine-binding aptamers with micromolar affinity, with Red.1 aptamer having a Kd of 7.9 ± 3.6 µM. Truncation improved the binding affinity to 5.2 ± 2.7 µM, and from this sequence, a Spiegelmer with improved nuclease resistance was created with a Kd of 2.0 ± 0.8 µM. This l-serine-binding Spiegelmer has the affinity and stability to be incorporated into aptamer-based biosensors for agricultural applications.

Pharmacokinetics, pharmacodynamics and safety of aptamers.

Aptamers are synthetic molecules structured as single-stranded DNA or RNA oligonucleotides that can be designed to mimic the functional properties of monoclonal antibodies. They bind to the target molecules (typically soluble or cell-bound proteins) with high affinity (with picomolar to low nanomolar range) and specificity, and therefore can be an alternative to therapeutic antibodies or peptide ligands. This paper reviews published data regarding pharmacokinetics, pharmacodynamics and safety of aptamers from preclinical and clinical studies. Aptamers have been developed for the treatment of a variety of diseases, including cancer, macular degeneration,g cardiovascular disease, diabetes and anaemia of chronic diseases. There are several preclinical studies with unmodified aptamers, but the vast majority of aptamer trials in humans have been conducted with modified aptamers, because unmodified aptamers demonstrate metabolic instability, as well as rapid renal filtration and elimination. Various strategies have been developed to improve the pharmacokinetic profile of aptamers. Aside from chemical modification of nucleotides in order to stabilize them against nuclease degradation, the main modification to extend the half-life is pegylation. Therefore, the process of pegylation as well as its benefits and possible shortcomings will briefly be discussed.

CXCL12/SDF-1-Dependent Retinal Migration of Endogenous Bone Marrow-Derived Stem Cells Improves Visual Function after Pharmacologically Induced Retinal Degeneration.

Mobilized bone marrow-derived stem cells (BMSC) have been discussed as an alternative strategy for endogenous repair. Thereby, different approaches for BMSC mobilization have been pursued. Herein, the role of a newly discovered oligonucleotide for retinal homing and regeneration capability of BMSCs was investigated in the sodium iodate (NaIO3) model of retinal degeneration. Mobilization was achieved in GFP-chimera with NOX-A12, a CXC-motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF-1)-neutralizing L-aptamer. BMSC homing was directed by intravitreal SDF-1 injection. Visual acuity was measured using the optokinetic reflex. Paraffin cross sections were stained with hematoxylin and eosin for retinal thickness measurements. Immunohistochemistry was performed to investigate the expression of cell-specific markers after mobilization. A single dose of NOX-A12 induced significant mobilization of GFP+ cells which were found in all layers within the degenerating retina. An additional intravitreal injection of SDF-1 increased migration towards the site of injury. Thereby, the number of BMSCs (Sca-1+) found in the damaged retina increased whereas a decrease of activated microglia (Iba-1+) was found. The mobilization led to significantly increased visual acuity. However, no significant changes in retinal thickness or differentiation towards retinal cell types were detected. Systemic mobilization by a single dose of NOX-A12 showed increased homing of BMSCs into the degenerated retina, which was associated with improved visual function when injection of SDF-1 was additionally performed. The redistribution of the cells to the site of injury combined with their observed beneficial effects support the endogenous therapeutic strategy for retinal repair.

Applications of nucleoside-based molecular probes for the in vivo assessment of tumour biochemistry using positron emission tomography (PET)

Positron emission tomography (PET) is a non-invasive nuclear imaging technique. In PET, radiolabelled molecules decay by positron emission. The gamma rays resulting from positron annihilation are detected in coincidence and mapped to produce three dimensional images of radiotracer distribution in the body. Molecular imaging with PET refers to the use of positron-emitting biomolecules that are highly specific substrates for target enzymes, transport proteins or receptor proteins. Molecular imaging with PET produces spatial and temporal maps of the target-related processes. Molecular imaging is an important analytical tool in diagnostic medical imaging, therapy monitoring and the development of new drugs. Molecular imaging has its roots in molecular biology. Originally, molecular biology meant the biology of gene expression, but now molecular biology broadly encompasses the macromolecular biology and biochemistry of proteins, complex carbohydrates and nucleic acids. To date, molecular imaging has focused primarily on proteins, with emphasis on monoclonal antibodies and their derivative forms, small-molecule enzyme substrates and components of cell membranes, including transporters and transmembrane signalling elements. This overview provides an introduction to nucleosides, nucleotides and nucleic acids in the context of molecular imaging.

A tomografia por emissão de pósitrons (TEP) é uma técnica de imagem não invasiva da medicina nuclear. A TEP utiliza moléculas marcadas com emissores de radiação beta positiva (pósitrons). As radiações gama medidas que resultam do aniquilamento dos pósitrons são detectadas por um sistema de coincidência e mapeadas para produzir uma imagem tridimensional da distribuição do radiotraçador no corpo. A imagem molecular com TEP refere-se ao uso de biomoléculas marcadas com emissor de pósitron que são substratos altamente específicos para alvos como enzimas, proteínas transportadoras ou receptores protéicos. A imagem molecular com TEP produz mapas espaciais e temporais de alvos que estejam sendo avaliados. A imagem molecular é uma importante ferramenta analítica no diagnóstico por imagem em medicina, no monitoramento de terapia e no desenvolvimento de novas drogas. A imagem molecular tem seus fundamentos na biologia molecular. Originalmente, a biologia molecular significava a biologia da expressão gênica, mas atualmente a biologia molecular envolve amplamente a biologia de macromoléculas, a bioquímica de proteínas, carboidratos complexos e ácidos nucléicos. A imagem molecular tem priorizado as proteínas, com ênfase nos anticorpos monoclonais e suas formas derivadas, substratos enzimáticos para pequenas moléculas e componentes de membranas celulares, incluindo os transportadores e elementos envolvidos com a sinalização trans-membrana.