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

Abstract

Rikenella microfusus is an essential intestinal probiotic with great potential. The latest research shows that imbalance in the intestinal flora are related to the occurrence of various diseases, such as intestinal diseases, immune diseases, and metabolic diseases. Rikenella may be a target or biomarker for some diseases, providing a new possibility for preventing and treating these diseases by monitoring and optimizing the abundance of Rikenella in the intestine. However, the current monitoring methods have disadvantages, such as long detection times, complicated operations, and high costs, which seriously limit the possibility of clinical application of microbiome-based treatment options. Therefore, the intention of this study was to evolve an enriched aptamer library to be used for specific labeling of R. microfusus, allowing rapid and low-cost detection methods and, ultimately the construction of aptamer-based biosensors. In this study, we used Rikenella as the target bacterium for an in vitro whole Cell-SELEX (Systematic Evolution of Ligands by EXponential Enrichment) to evolve and enrich specific DNA oligonucleotide aptamers. Five other prominent anaerobic gut bacteria were included in this process for counterselection and served as control cells. The aptamer library R.m-R13 was evolved with high specificity and strong affinity (K_d = 9.597 nM after 13 rounds of selection). With this enriched aptamer library, R. microfusus could efficiently be discriminated from the control bacteria in complex mixtures using different analysis techniques, including fluorescence microscopy or fluorometric suspension assays, and even in human stool samples. These preliminary results open new avenues toward the development of aptamer-based microbiome bio-sensing applications for fast and reliable monitoring of R. microfusus.

Keywords: Cell-SELEX; DNA aptamer; Rikenella microfusus; biosensor; in vitro diagnostic.

Figure 1

SELEX is a directed evolution process performed completely in the laboratory leading to oligonucleotide DNA or RNA molecules (aptamers) with binding affinity to target structures. In principle, a (commercial) starting library of up to 10¹⁴ individual sequences is used for binding to the desired target. In an iterative process of binding, washing off non-binders, harvesting of binders, and their amplification by PCR, the affinity and specificity of the aptamers will increase (or “evolve”). A prerequisite is a certain selection pressure toward better binding, which can gain stringency by reducing the amount of aptamers provided, increasing the harshness of washing, and using so-called counter-selection steps using non-specific targets like other gut bacteria and/or non-aptamer nucleic acids as well as blocking proteins like casein. Here, the selection pressure and thus the efficiency of evolution were enhanced by incubating the initial library with a mixture of control bacteria, including A. muciniphila, A. stercoricanis, B. producta, R. intestinalis, and P. distasonis, during the initial rounds of selection. Counter-SELEX was used to eliminate aptamers unbound to target bacteria. Subsequently, the aptamers obtained in the previous evolution round were incubated with the target bacterium, R. microfusus, for target SELEX. After repeating several rounds of screening/selection, we obtained aptamer eluates to obtain new pools of enriched DNA by PCR amplification. Ultimately, we analyzed the enriched aptamer libraries we obtained from those specific binding target cells in various ways, including fluorescence analysis, fluorescence microscopy observation, and qPCR quantitative analysis.

Figure 2

(a) Affinity and (b) specificity assay for R. microfusus, using the aptamer library R.m-R13. The specificity of the enriched aptamer library obtained in rounds 11–13 increased progressively compared to the mixture of the other five intestinal bacteria B. producta, A. muciniphila, R. intestinalis, A. stercoricanis, and P. distasonis. All experiments were performed using five pmol aptamer and 10⁸ cells, and fluorescence intensity was measured at 635 nm excitation and 670 nm emission. All experiments were performed in three sessions (N = 3). (c) Melting curves of the prominent temperature peaks, including S_Tm at the start and E_Tm at the end of SELEX; (d) peak shift analyses for S_Tm and E_Tm, and linear regression fitted to ddRn/dT (fluorescence change/temperature change) with different SELEX rounds.

Figure 3

(a) Determination of the dissociation constant for the aptamer library R.m-R13 with a K_d of 9.597 nM and a deviation of 0.9839 in the coefficient of determination R². (b) Fluorescence microscopy observations of the aptamer library R.m-R13 bound to R. microfusus, B. producta, A. muciniphila, R. intestinalis, A. stercoricanis, and P. distasonis. Cy5-labeled R.m-R13 displayed a strong fluorescent signal bound to R. microfusus, whereas the other five bacteria as controls delivered, as expected, no signals. (c) Quantification of the aptamer library R.m-R13 on R. microfusus in a mixture that included B. producta, A. muciniphila, R. intestinalis, A. stercoricanis, and P. distasonis, mixed in different proportions at the same OD. All experiments were performed using five pmol aptamers in 10⁸ cells, and all experiments were performed in three runs (N = 3). p values < 0.05 were considered significant. * denotes p < 0.05, *** denotes p < 0.001.

Figure 4

(a) Sensitivity assay of the aptamer library R.m-R13. In a 1 mL reaction system containing five pmol of the aptamer library, R.m-R13 showed a significant positive correlation between the number of bacteria and fluorescence intensity for R. microfusus numbers greater than 10² (R² = 0.9889), and therefore, the minimum detection limit for the aptamer library was determined to be 10² cells/mL. (b) Comparison of Rikenella abundance measurements in fecal samples from the aptamer library R.m-R13 and 16s rRNA NGS (see Tables S2 and S3, Supplementary Material). “NGS values” represent the actual Rikenella content in fecal bacteria. The “Aptamer value” represents the amount of Rikenella in fecal bacteria determined using the aptamer library R.m-R13. Error bars indicate the standard deviations of the experiments conducted in triplicate.