Development of a whole-cell SELEX process to select species-specific aptamers against Aspergillus niger

Abstract

Background: Spores produced by the filamentous fungus Aspergillus niger are abundant in a variety of environments. The proliferation of this fungus in indoor environments has been associated to health risks and its conidia can cause allergic reaction and severe invasive disease in animals and humans. Therefore, the detection and monitoring of Aspergillus conidia is of utmost importance to prevent serious fungal infections and contaminations. Among others, aptamers could serve as biosensors for the specific detection of fungal spores.

Results: In this study, DNA aptamers specific to conidia of A. niger were developed by optimizing a whole-cell SELEX approach. Three whole-cells SELEX experiments were performed in parallel with similar conditions. Quantification of recovered ssDNA and melting curve analyses were applied to monitor the ongoing SELEX process. Next-generation sequencing was performed on selected recovered ssDNA pools, allowing the identification of DNA aptamers which bind with high affinity to the target cells. The developed aptamers were shown to be species-specific, being able to bind to A. niger but not to A. tubingensis or to A. nidulans. The binding affinity of two aptamers (AN01-R9-006 and AN02-R9-185) was measured to be 58.97 nM and 138.71 nM, respectively, which is in the range of previously developed aptamers.

Conclusions: This study demonstrates that species-specific aptamers can be successfully developed via whole-cell SELEX to distinguish different Aspergillus species and opens up new opportunities in the field of diagnostics of fungal infections.

Keywords: Aspergillosis; Biosensors; DNA aptamers; Fungal conidia; Spores.

Fig. 1

Schematic illustration of the whole-cell SELEX process applied to select aptamers specific to A. niger conidia. Nine consecutive rounds and three SELEX experiments were performed. Counter-selection with A. tubingensis was introduced after round 4. This figure was partly created with https://www.biorender.com/

Fig. 2

Exemplary results from test-PCRs dnc1 (A) and dnc2 (B) performed on recovered ssDNA to determine the optimal number of cycles for subsequent amplification. In A, amplification of ssDNA recovered in round 3 of SELEX-3 (R3_SELEX) is shown alongside with the negative control (R3_NC) and the non-template control (NTC). The circle highlights the number of cycles (18) at which the peak of the fluorescence signal is reached while the three arrows point at the number of cycles (15, 16 and 17) before the peak which were chosen for testing in dnc2. In dnc1 the fluorescence signal was normalized to 0. In Figure B, L: ladder, 15, 16 and 17: PCR product after 15, 16 and 17 cycles

Fig. 3

Quantification by qPCR of the ssDNA recovered after each round in the three SELEX experiments (A: SELEX-1, B: SELEX-2 and C: SELEX-3). R2 to R9-T refer to the specific rounds of SELEX-1, SELEX-2 and SELEX-3 described in Tables 1, 2 and 3, respectively. Error bars indicate min. and max. values

Fig. 4

Melting curves of the ssDNA recovered after each round in the three SELEX experiments (A : SELEX-1, B : SELEX-2 and C: SELEX-3). R2 to R9-T refer to the specific rounds of SELEX-1, SELEX-2 and SELEX-3 described in Tables 1, 2 and 3, respectively. Melting curve of 10⁵ molecules of the original ssDNA library (ssDNA 10^5) was performed as comparison

Fig. 5

Total count of individual sequences over the rounds shown in percentage. Sequencing was performed on ssDNA pools recovered after rounds 2, 6, 7, 8, 9-N and 9-T of SELEX-1 and SELEX-2 and after all rounds of SELEX-3. Additionally, the initial ssDNA library was sequenced to check for biases. R2 to R9-T refer to the specific rounds of SELEX-1, SELEX-2 and SELEX-3 described in Tables 1, 2 and 3, respectively

Fig. 6

First screening of FAM-labelled candidate aptamers. Recovered ssDNA after binding assays with A. niger conidia was quantified by qPCR and is shown in molecules/µL. Samples were measured in duplicates. The mean value between biological and technical replicates is displayed after subtracting the mean value of the blank samples (only aptamers, no conidia). Error bars represent max. and min. values. Asterisks indicate aptamers showing significantly different binding (p-value < 0.05) compared to the negative controls (ssDNA library and BA-NC-1)

Fig. 7

Second screening of FAM-labelled and unlabeled candidate aptamers. Recovered ssDNA after binding assays with A. niger conidia was quantified by qPCR and is shown in molecules/µL. Samples were measured in technical duplicates. The mean value between biological and technical replicates is displayed after subtracting the mean value of the blank samples (only aptamers, no conidia). Error bars represent max. and min. values. Asterisks indicate aptamers showing significantly different binding (p-value < 0.05) between unlabelled and labelled version

Fig. 8

Specificity evaluation of selected aptamers by means of qPCR (A) or fluorescence (B) measurements. A: recovered ssDNA after binding assays with A. niger, A. tubingensis and A. nidulans conidia was quantified by qPCR and is shown in molecules/µL. Samples were measured in technical duplicates. The mean value between biological and technical replicates is displayed after subtracting the mean value of the blank samples (only aptamers, no conidia). The number of biological and technical replicates performed is indicated in white in each bar. Error bars represent max. and min. values. The predicted secondary structure of each aptamer was obtained with RNAFold and is shown on top of the figure. B: confirmation of the species-specificity of three selected aptamers by fluorescent measurements upon elution. Fluorescence of the recovered and eluted ssDNA after binding assays with A. niger, A. tubingensis and A. nidulans conidia was measured at a Tecan Spark reader. Samples were measured in duplicates. The mean value between biological and technical replicates is displayed after subtracting the mean value of the blank samples (only conidia, no aptamer). Error bars represent max. and min. values. Asterisks indicate aptamers showing significantly different binding (p-value < 0.05) between different strains

Fig. 9

Binding affinity of aptamers AN01-R9-006 (A) and AN02-R9-185 (B) to A. niger conidia. Single measurements are indicated by blue crosses and the corresponding fitted curve is shown as a black line. The red dotted line corresponds to the K_D value