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

Abstract

Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes.

Robust Fluorometric Aptamer Assay for Direct and Rapid Detection of Clinical Isolates of Candida spec.

“…With the finding that polyclonal aptamer libraries originating directly from SELEX processes may outperform the individual aptamers [ 52 ] selected from these libraries, we introduced such enriched or “polyclonal” aptamer libraries as valuable tools for the fluorescent labeling of target structures and thus the specific quantification of a series of microbial symbionts and pathobionts [ 52 , 53 , 54 , 55 , 56 , 57 ]. Moreover, the functionality of this concept was further demonstrated with different tissues of plant roots [ 58 ] and by using specific enriched libraries for the construction of electronic gFET-based biosensors to measure pre-diabetes-related biomarkers [ 59 ].…”

Polyclonal Aptamer Libraries from a FluRoot-SELEX for the Specific Labeling of the Apical and Elongation/Differentiation Zones of Arabidopsis thaliana Roots

“…Fluorescence microscopy provided the opportunity to visualize and analyze the specific target binding sites of both the final aptamer libraries. Therefore, 5 pmol from each aptamer library was activated according to the prior description and incubated with either the root center or tip for 60 min at room temperature in darkened conditions on a rotator [ 10 , 12 , 13 , 14 , 15 , 16 , 17 ]. Thereafter, the root zone segments were washed two times using 100 µL DPBS and were prepared on a slide with a glass cover.…”

“…Nowadays, adapted SELEX processes such as Capture-SELEX or FluMag-SELEX enable the selection of aptamers for small solute molecules [ 6 ], chemical compounds such as metal ions [ 7 ], as well as proteins and their cofactors [ 8 , 9 , 10 ]. To obtain aptamers that are specific towards whole cells and certain microorganisms, Cell-SELEX and FluCell-SELEX have been developed to target intact living cells such as the pathogenic bacterium Pseudomonas aeruginosa or different mammalian cells, such as cancer cells, without knowing the exact membrane target in advance [ 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Even against complex consortia of cells such as tumors, specialized technologies allow for the selection of highly specific aptamers to work on pathological tissues [ 18 , 19 ].…”

Enriched Aptamer Libraries in Fluorescence-Based Assays for Rikenella microfusus-Specific Gut Microbiome Analyses

“…In our previous studies, we have facilitated our real-time monitoring of the screening/selection process by Cy5 fluorescent-labeled DNA aptamers and direct measurement of aptamer binding to target cells by changes in fluorescence intensities on the cells. This technique we introduced as the FluCell-SELEX [ 40 , 41 , 42 , 43 , 44 ]. In order to boost the SELEX screening pressure and improve stringency, a mixture of five other intestinal bacteria was used in a counter-selection, including A. muciniphila , A. stercoricanis , B. producta , R. intestinalis, and P. distasonis in this study ( Figure 1 ).…”