Fig 1.
Selection strategy used during SELEX to enrich for multiple, site-specific DNA aptamers.
Table 1.
Aptamer sequences and their variations used in this study.
Fig 2.
Monitoring of the progress of selection through SELEX by qPCR using samples of ssDNA eluted from the surfaces of beads used during SELEX.
A) Absolute DNA quantity obtained by qPCR for negative and positive selection steps at each round of selection. B) Amplification curves obtained for different selection rounds analysed by qPCR. C) Melting curves obtained for different selection rounds analysed by qPCR.
Fig 3.
Bioinformatic analysis of sequence enrichment as SELEX progressed, showing the groups of sequences anticipated from the SELEX procedure in this study.
Fig 4.
Screening of the ability of selected aptamer sequences to bind to the hCG target, evaluated using a variety of binding assays.
A) Initial screening of aptamer candidates′ ability to bind unimmobilised hCG assessed by EMSA. Representative GelRed stained gel images are shown with or without hCG incubation. The difference in pixel intensity of the DNA bands for each sequence was calculated in the presence and absence of hCG using ImageJ analysis software. Annotations: †- changes in band intensity in the presence of the target ≥ 10% are considered significant. B) qPCR-based quantification of aptamer sequences binding to beads coated either with hCG or the control protein, HSA. Assays were performed in triplicate, with all three values shown. Annotations: †—Sequences exhibiting significantly higher quantities of DNA bound to hCG-modified beads, compared to HSA-modified beads (two-tailed t-tests, p ≤ 0.05). *—Sequences exhibiting significantly higher binding to hCG-modified beads compared to the Lib_1 control sequence (one-way ANOVA results annotated in Fig; sequences identified using subsequent Tukey HSD post-hoc test, p ≤ 0.05). C) Spectrophotometric ELONA assay of biotinylated aptamer sequences binding to beads coated either with hCG or the control protein, BSA. Following exposure of the aptamers to hCG- or BSA-coated magnetic beads, bound aptamers were subsequently quantified using the streptavidin-HRP and TMB reporter system. Assays were performed in triplicate, with all three values shown. Annotations: †—Sequences exhibiting significantly higher quantities of DNA bound to hCG-modified beads, compared to HSA-modified beads (two-tailed t-tests, p ≤ 0.05). ‡—Sequences exhibiting suggestively higher quantities of DNA bound to hCG-modified beads, compared to HSA-modified beads (two-tailed t-test, p ≤ 0.1). *—Sequences exhibiting significantly higher binding to hCG-modified beads compared to “No DNA” control samples (one-way ANOVA results annotated in Fig; sequences identified using subsequent Tukey HSD post-hoc test, p ≤ 0.05). D) hCG Coated Paper-format ELONA screening of aptamer sequences. hCG was covalently attached to the paper surface within each barrier and exposed to biotinylated sequences. Bound sequences were subsequently quantified using an anti-biotin antibody and subsequent quantification using a secondary antibody-HRP and TMB system. Representative images captured of the colorimetric signal generated for different aptamer sequences tested by indirect paper-based ELONA (top panel). The signal intensity for each sample was quantified by ImageJ and is presented as a univariate plot (bottom panel). Annotations: *—Sequences exhibiting significantly higher binding to hCG-modified wells compared to R4_1 control samples (one-way ANOVA results annotated in Fig; sequences identified using subsequent Tukey HSD post-hoc test, p ≤ 0.05).
Fig 5.
Competitive assays used to elucidate aptamer candidates′ target epitope specificity.
A) Evaluation of conditions required to capture hCG when using aptamer sequence R4_64. Wells were coated with streptavidin, subsequently modified with biotinylated aptamer sequences and exposed to hCG. hCG captured by this system was quantified using the anti-α subunit antibody, subsequently using a HRP-conjugated secondary antibody and TMB to quantify the presence of the anti-α antibody. Different aptamer conditions were compared to control sequences which lacked any biotin modification (unmodified full-length R4_64) and any capturing DNA (no DNA). Triplicate measurements were generated and shown. Annotations: *—Sequences exhibiting significantly higher binding to hCG-modified beads compared to “No DNA” control samples (one-way ANOVA results annotated in Fig; sequences identified using subsequent Tukey HSD post-hoc test, p ≤ 0.05). B) Screening of aptamer sequences capable of binding to hCG sites that do not compete with R4_64′s binding. Full-length R4_64, folded in the presence of the complementary blocking oligonucleotides, was immobilised onto streptavidin-coated magnetic beads through its attached biotin moiety. Beads were exposed to hCG and subsequently, full-length, oligomer-blocked aptamer candidates. Following DNA elution, aptamers bound to the beads were quantified by SYBR qPCR. Triplicate measurements were made and shown. Annotations: *—Sequences exhibiting significantly higher binding to hCG captured by R4_64, compared to the “None” control samples (one-way ANOVA results annotated in Fig; sequences identified using subsequent Tukey HSD post-hoc test, p ≤ 0.05).