Polyclonal Aptamers for Specific Fluorescence Labeling and Quantification of the Health Relevant Human Gut Bacterium Parabacteroides distasonis

Abstract

Single-stranded DNA aptamers as affinity molecules for the rapid, reliable detection of intestinal bacteria are of particular interest to equip health systems with novel robust and cheap diagnostic tools for monitoring the success of supplementation strategies with selected probiotic gut bacteria in the fight against major widespread threats, such as obesity and neurodegenerative diseases. The human gut bacterium Parabacteroides distasonis (P. distasonis) is positively associated with diseases such as obesity, non-alcoholic fatty liver disease and multiple sclerosis with reduced cell counts in these diseases and is thus a promising potential probiotic bacterium for future microbial supplementation. In this paper we report on the evolution of a specific polyclonal aptamer library by the fluorescence based FluCell-SELEX directed against whole cells of P. distasonis that specifically and efficiently binds and labels P. distasonis. The aptamer library showed high binding affinity and was suited to quantitatively discriminate P. distasonis from other prominent gut bacteria also in mixtures. We believe that this library against a promising probiotic bacterium as a prototype may open new routes towards the development of novel biosensors for the easy and efficient quantitative monitoring of microbial abundance in human microbiomes in general.

A Polyclonal Aptamer Library for the Specific Binding of the Gut Bacterium Roseburia intestinalis in Mixtures with Other Gut Microbiome Bacteria and Human Stool Samples

“…The polyclonal libraries presented in this study originating from the FluCell-SELEX against R. intestinalis were characterized and found to increase their specificity up to the final round, 7_2. As described for the libraries from previous projects [ 31 , 35 , 36 , 40 ], this library Ri 7_2 was suitable for the labeling of R. intestinalis in fluorescence-based microtiter plate assays and for fluorescence microscopy. The measurements were also performed as microbiome samples derived from the stool samples of two human probands and compared to measurements of sequence abundance via NGS.…”

“…Electronic biosensors are easy to handle, often portable, and, in principle, can analyze in real-time, making them suited for measuring biomarkers [ 30 ]. In recent years, aptamer-based biosensors have received much attention for their excellent performance in terms of sensitivity, stability and selectivity [ 31 , 32 ]. Specific aptamers usually originate from libraries of single-stranded RNA or single-stranded DNA fragments obtained by the systematic evolution of ligands by exponential enrichment (SELEX) technology.…”

“…Specific aptamers usually originate from libraries of single-stranded RNA or single-stranded DNA fragments obtained by the systematic evolution of ligands by exponential enrichment (SELEX) technology. The aptamers can be evolved towards a high specificity and affinity for target molecules, such as proteins, amino acids, viruses, pathogens and higher cells and tissues [ 31 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. Initial random libraries consist of 10 12 –10 16 single-stranded deoxyribonucleic acid (ssDNA) molecules and each sequence contains fixed primer binding sites and a random region in the center of the molecule.…”

“…Initial random libraries consist of 10 12 –10 16 single-stranded deoxyribonucleic acid (ssDNA) molecules and each sequence contains fixed primer binding sites and a random region in the center of the molecule. After incubation of the random library with the target cells during the screening process, the DNA sequences bound to the target cells are amplified by PCR, and the next round of screening is performed [ 31 , 36 ]. Based on our previous studies, we systematically evolved a library of Cyanine 5 (Cy5) fluorescently labeled DNA aptamers that bind specifically to the target cells using a technique called FluCell-SELEX.…”

A Polyclonal SELEX Aptamer Library Allows Differentiation of Candida albicans, C. auris and C. parapsilosis Cells from Human Dermal Fibroblasts

“…In an iterative selection process performed completely in vitro, termed Systematic Evolution of Ligands by EXponential enrichment (SELEX), high-affinity aptamers can be evolved and isolated from large random sequence libraries ( Figure 1 ). There aptamers can be selected against an enormous variety of different target structures ranging from small molecules such as metal ions [ 4 ], proteins [ 5 ], to cells and microorganisms [ 6 , 7 , 8 , 9 ] and even to complex targets such as cancerous tissues [ 10 , 11 ]. This directed selection process has continuously been improved and modified to create more powerful, robust and specialized strategies that allow the selection of aptamers with specific binding properties for different targets in numerous applications.…”