FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium Akkermansia muciniphila.

Based on their unique properties, oligonucleotide aptamers have been named a gift of biological chemistry to life science. We report the development of DNA aptamers as the first high-affinity binding molecules available for fast and rapid labeling of the human gut bacterium Akkermansia muciniphila with a certain impact on Alzheimer´s disease. Fast and reliable analyses of the composition of microbiomes is an emerging field in microbiology. We describe the molecular evolution and biochemical characterization of a specific aptamer library by a FluCell-SELEX and the characterization of specific molecules from the library by bioinformatics. The aptamer AKK13.1 exerted universal applicability in different analysis techniques in modern microbiology, including fluorimetry, confocal laser scanning microscopy and flow cytometry. It was also functional as a specific binding entity hybridized to anchor primers chemically coupled via acrydite-modification to the surface of a polyacrylamide-hydrogel, which can be prototypically used for the construction of affinity surfaces in sensor chips. Together, the performance and methodological flexibility of the aptamers presented here may open new routes not only to develop novel Akkermansia-specific assays for clinical microbiology and the analyses of human stool samples but may also be an excellent starting point for the construction of novel electronic biosensors.

Diffusion-controlled release of the theranostic protein-photosensitizer Azulitox from composite of Fmoc-Phenylalanine Fibrils encapsulated with BSA hydrogels.

Hydrogels offer a promising potential for the encapsulation and regulated release of drugs due to their biocompatibility and their tunable properties as materials. Only a limited number of systems and procedures enable the encapsulation of sensitive proteins. N-terminally fmoc-protected phenylalanine has been shown to self-assemble into a transparent, stable hydrogel It can be considered a supergelator due to the low amount of monomers necessary for hydrogelation (0.1% w/v), making it a good candidate for the encapsulation and stabilization of sensitive proteins. However, application options for this hydrogel are rather limited to those of many other fibril-based materials due to its intrinsic lack of mechanical strength and high susceptibility to changes in environmental conditions. Here, we demonstrate that the stability of a fibrillary system and the resulting release of the protein-photosensitizer Azulitox can be increased by combining the hydrogel with a tightly cross-linked BSA hydrogel. Azulitox is known to display cell-penetrating properties, anti-proliferative activity and has a distinctive fluorescence. Confocal microscopy and fluorescence measurements verified the maintenance of all essential functions of the encapsulated protein. In contrast, the combination of fibrillary and protein hydrogel resulted in a significant stabilization of the matrix and an adjustable release pattern for encapsulated protein.

Azulitox-A Pseudomonas aeruginosa P28-Derived Cancer-Cell-Specific Protein Photosensitizer.

Azulitox as a new fusion polypeptide with cancer cell specificity and phototoxicity was generated and is composed of a photosensitizer domain and the cell-penetrating peptide P28. The photosensitizer domain (EcFbFP) was derived from a bacterial blue-light receptor, which belongs to the family of light-oxygen-voltage proteins and produces reactive oxygen species (ROS) upon excitation. P28 is derived from the cupredoxin protein azurin that is known to specifically penetrate cancer cells and bind to the tumor suppressor protein p53. We show that the P28 domain specifically directs and translocates the fused photosensitizer into cancer cells. Under blue-light illumination, Azulitox significantly induced cytotoxicity. Compared to the extracellular application of EcFbFP, Azulitox caused death to about 90% of cells, as monitored by flow cytometry, which also directly correlated with the amount of ROS produced in the cells. Azulitox may open new avenues toward targeted polypeptide-photosensitizer-based photodynamic therapies with reduced systemic toxicity compared to conventional photosensitizers.