Immobilization-Free Determination of Dissociation Constants Independent of Ligand Size Using MicroScale Thermophoresis.

The quantitative characterization of aptamer-ligand interactions is an important step in the aptamer development process. However, certain pitfalls impede KD determination, especially when working with small molecule ligands. These include altered binding behavior caused by ligand immobilization. Further, the compulsory requirement for major differences in size between the bound and unbound state makes small molecule ligands ineligible for separation-based methods. MicroScale Thermophoresis circumvents such limitations as binding is accurately quantified with both binding partners free in solution and independent of ligand size. In this chapter, we present a protocol for the characterization of a DNA aptamer binding to its small molecule ligand daunorubicin.

Aptamer-Based Sandwich Assay Formats for Detection and Discrimination of Human High- and Low-Molecular-Weight uPA for Cancer Prognosis and Diagnosis.

Urokinase-type plasminogen activator (urokinase, uPA) is a frequently discussed biomarker for prognosis, diagnosis, and recurrence of cancer. In a previous study, we developed ssDNA aptamers that bind to different forms of human urokinase, which are therefore assumed to have different binding regions. In this study, we demonstrate the development of aptamer-based sandwich assays that use different combinations of these aptamers to detect high molecular weight- (HMW-) uPA in a micro titer plate format. By combining aptamers and antibodies, it was possible to distinguish between HMW-uPA and low molecular weight- (LMW-) uPA. For the best performing aptamer combination, we calculated the limit of detection (LOD) and limit of quantification (LOQ) in spiked buffer and urine samples with an LOD up to 50 ng/mL and 138 ng/mL, respectively. To show the specificity and sequence dependence of the reporter aptamer uPAapt-02-FR, we have identified key nucleotides within the sequence that are important for specific folding and binding to uPA using a fluorescent dye-linked aptamer assay (FLAA). Since uPA is a much-discussed marker for prognosis and diagnosis in various types of cancers, these aptamers and their use in a micro titer plate assay format represent a novel, promising tool for the detection of uPA and for possible diagnostic applications.

Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA.

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a KD of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a KD of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.

The use of high-affinity polyhistidine binders as masking probes for the selection of an NDM-1 specific aptamer.

The emergence of carbapenemase-producing multi-drug resistant Enterobacteriaceae poses a dramatic, world-wide health risk. Limited treatment options and a lack of easy-to-use methods for the detection of infections with multi-drug resistant bacteria leave the health-care system with a fast-growing challenge. Aptamers are single stranded DNA or RNA molecules that bind to their targets with high affinity and specificity and can therefore serve as outstanding detection probes. However, an effective aptamer selection process is often hampered by non-specific binding. When selections are carried out against recombinant proteins, purification tags (e.g. polyhistidine) serve as attractive side targets, which may impede protein target binding. In this study, aptamer selection was carried out against N-terminally hexa-histidine tagged New Delhi metallo-ß-lactamase 1. After 14 selection rounds binding to polyhistidine was detected rather than to New Delhi metallo-ß-lactamase 1. Hence, the selection strategy was changed. As one aptamer candidate showed remarkable binding affinity to polyhistidine, it was used as a masking probe and selection was restarted from selection round 10. Finally, after three consecutive selection rounds, an aptamer with specific binding properties to New Delhi metallo-ß-lactamase 1 was identified. This aptamer may serve as a much-needed detection probe for New Delhi metallo-ß-lactamase 1 expressing Enterobacteriaceae.

Clostridium ramosum regulates enterochromaffin cell development and serotonin release.

Peripheral serotonin (5-hydroxytryptamine: 5-HT) synthesized in the intestine by enterochromaffin cells (ECs), plays an important role in the regulation of peristaltic of the gut, epithelial secretion and promotes the development and maintenance of the enteric neurons. Recent studies showed that the indigenous gut microbiota modulates 5-HT signalling and that ECs use sensory receptors to detect dietary and microbiota-derived signals from the lumen to subsequently transduce the information to the nervous system. We hypothesized that Clostridium ramosum by increasing gut 5-HT availability consequently contributes to high-fat diet-induced obesity. Using germ-free mice and mice monoassociated with C. ramosum, intestinal cell lines and mouse organoids, we demonstrated that bacterial cell components stimulate host 5-HT secretion and program the differentiation of colonic intestinal stem progenitors toward the secretory 5-HT-producing lineage. An elevated 5-HT level regulates the expression of major proteins involved in intestinal fatty acid absorption in vitro, suggesting that the presence of C. ramosum in the gut promotes 5-HT secretion and thereby could facilitates intestinal lipid absorption and the development of obesity.