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Institute
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.
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 K-D of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a K-D 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.