Specific Aspects of SELEX Protocol: Different Approaches for ssDNA Generation

Abstract

Background: Synthetic DNA aptamers are a class of molecules with potential applications in medicine, serving as molecular sensors or ligands for targeted drug delivery. Systematic evolution of ligands by exponential enrichment (SELEX) is a technology for selecting functional aptamers that was first reported three decades ago and has been actively developed since. SELEX involves multiple iterations of two fundamental steps: (i) target affinity-based partitioning of aptamers from a random library and (ii) amplification of selected aptamers by PCR, followed by isolation of single-stranded DNA (ssDNA). SELEX protocols have diversified considerably, with numerous variations possible for each step. This heterogeneity makes it challenging to identify optimal methods. Comparative analysis of different approaches for the major stages of SELEX is therefore of considerable practical importance.

Methods: Four widely used methods for ssDNA generation were performed in parallel: (a) PCR followed by digestion of the antisense strand with exonuclease lambda, (b) PCR with an extended primer followed by size-dependent strand separation using denaturing PAGE, (c) asymmetric PCR, and (d) asymmetric PCR with a primer-blocker.

Results: The specificity, efficiency, reproducibility, and duration of each method were compared.

Conclusions: Asymmetric PCR with a primer-blocker yielded the most favorable results.

Keywords: PCR; SELEX; aptamer; asymmetric PCR; single-stranded DNA.

Figure 1

Scheme of ssDNA generation methods.

Figure 2

The impact of RV(5′-ph) primer quality on the efficiency of exonuclease digestion in the PCR-lambda method was investigated. Four parallel PCR reactions were performed, differing only in the source of the RV(5′-ph) primer. The amplification products were purified, and 1 µg of dsDNA from each reaction was digested by lambda exonuclease for 30 min. The products were then separated on a 3% agarose gel Line 1—Step50 plus DNA marker and for lines 2 and 7, ssDNA library used for PCR with a size equal to the expected ssDNA product of exonuclease reaction. The lines 3, 4, 5 and 6 are the results of experiments carried out with different RV(5′-ph). In each pair of lines: a—dsDNA PCR product (≈20 ng); b—product of exonuclease reaction (≈60 ng) made from a mixture of dsDNA and ssDNA.

Figure 3

Electrophoresis in 6% PAGE of PBA-PCR amplification products. Lanes 1a, 2a, 3a and 4a correspond to 20, 22, 24 and 26 cycles of PCR with all primers: FW, RV(pA) and RV(bl) as shown in scheme (A). Lanes 1b, 2b, 3b and 4b correspond to 20, 22, 24 and 26 cycles of PCR with only FW and RV(bl) primers as shown in scheme (B).

Figure 4

Electrophoresis in 6% PAGE of amplification products obtained at consequent cycles by the four methods tested. (a) PCR-lambda; (b) PCR-long RV; (c) A-PCR; (d) PBA-PCR. Lines 1 and 8: DNA Ladder (GeneRuler 50 bp). Gels were imaged on an iBright FL1000 imaging system in fluorescent blot imaging mode using the FITC and Qdots 605 channels. Lines 2, 3, 4, 5, and 6 correspond to the PCR cycle numbers 5, 10, 15, 20 and 25 at which the amplification was completed. Line 7: template-free control. Double arrows indicate dsDNA products; single arrows indicate ssDNA products.

Figure 5

Principle of assessment of PCR specificity with ImageJ software. The total pixel value of the line was assumed to be 100%, and the area containing a specific product was estimated as a percentage.

Figure 6

Generation and separation of ssDNA by electrophoresis in 6% PAGE. The images show products generated by different methods from the same amount of ssDNA. In all cases, lines 1–3 contain the PCR product combined from 10 reactions, line 4 includes either Step50 plus DNA marker ((a,c,d) in PAGE) or ssDNA library ((b) in dPAGE). Bands containing ssDNA, indicated by frames, were excised for further purification.

Figure 7

Quantity ssDNA generated by four different methods from the same amount of ssDNA (3 × 10 PCR reactions). Products of PCR were pooled, ssDNA was purified by corresponding approaches and dissolved in 10 μL of water and 3 μL of ssDNA solution was loaded into denaturating gel for electrophoresis. (a) Fragment of denaturing 6% PAGE; (b) pixel density of bands quantified by ImageJ software. In both images, lines correspond to 100 ng library DNA (L), ssDNA generated by PCR-lambda (A), PCR-long RV (B), A-PCR (C) and PBA-PCR method (D).