Selection of aptamers for AMACR detection from DNA libraries with different primers

Abstract

In this work, we investigated aptamer selection against alpha-methylacyl-CoA racemase (AMACR) with three DNA libraries bearing different primers. Because of increased selection diversity, aptamers with varied structural properties, such as pre-folded, induced-folding, high and low primer-dependent conformations, were discovered. From the selection results, a dimeric aptamer was constructed and capable of detecting over-expression of AMACR in prostate cancer cell lines. In summary, this study demonstrates a library-based approach to obtain aptamers with different binding properties and provides distinct aptamers for flexible design of AMACR detection.

Scheme 1. Selection of anti-AMACR aptamers from three DNA libraries (N₃₀) with different primers by single-bead SELEX.

Fig. 1. Comparisons of (A) the CD spectra at a constant temperature (4 °C) and (B) the differential CD melting curve (−d[θ]_{275 nm}/dT vs. temperature) for Libraries A (black line, □), B (red line,△), and C (blue line, ○).

Fig. 2. Evolution of AMACR-specific ssDNA ligands of (A) Library A, (B) Library B, and (C) Library C during SELEX. The ratios of the amount of ssDNA molecules bound to AMACR-coated beads ([DNA]_A) to that bound to HSA-coated beads ([DNA]_H) were determined by qPCR.

Fig. 3. The GC content distributions of the randomized N₃₀ regions of aptamer candidates from (A) Library A, (B) Library B and (C) Library C. N_GC is the number of the aptamer with a specific GC content, and N_total is the total number of aptamer candidates compared.

Fig. 4. The CD spectra for (A) A24, (B) A38, (C) B4, (D) B38, (E) C14, and (F) C20 with (black line, □) and without (red line, △) AMACR.

Fig. 5. ELAA-based total binding assays for the binding affinity estimation of the anti-AMACR aptamers ((A) A24 and A38; (B) B4 and B38; (C) C14and C20). The AMACR concentration for microtiter plate's well coating was 0.2 μM.

Fig. 6. The proof-of-principle fluorescent ELAA-based AMACR detection using aptamers (A) A24, (B) A38, (C) B4, (D) B38, (E) C14, and (F) C20. The aptamer concentrations were 0.5 μM, and HSA was also assayed for specificity assessment.

Fig. 7. Comparisons of the binding characteristics of the as-selected and primer-truncated aptamers using the fluorescent ELAA method. (A) A38, (B) B4, and (C) C14.

Fig. 8. (A) ELAA-based total-binding assays for the binding affinity estimation of the dimeric, truncated C14 aptamer probe (DC14). The AMACR concentration for microtiter plate's well coating was 0.2 μM. (B) ELAA-based binding assays of cell lysates from E. coli with and without recombinant AMACR gene and two prostate cancer cell lines using DC14. The aptamer concentration was 0.2 μM.