In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

Abstract

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

Fig 1. Most abundant aptamer sequences in the selected aptamer pool.

Seven groups with 3–8 homologous sequences (#: number of homologous sequences) were identified among the sequenced aptamer clones. The representative aptamer clone of each group is shown. The specific primer binding sites at the 5’- and 3’-end of the aptamer clones are colored in red and blue, respectively.

Fig 2. Individual binding abilities of the most abundant aptamer sequences to Protein A.

The representative aptamer clones of the seven sequence groups were tested for their binding ability to Protein A in comparison to the selected aptamer pool and the unselected SELEX library. Bead-based binding assays were performed using Protein A/Strep-MB and fluorescein-labeled ssDNA. Target-bound aptamers were eluted and quantified.

Fig 3. Potential secondary structure of aptamer PA#2/8.

The primer binding sites (18 nt each) at the 5’- and 3’-end are highlighted in red and blue, respectively. The four G-stretches in the intern sequence region are highlighted in grey.

Fig 4. Binding curve of aptamer PA#2/8 obtained by bead-based binding assays.

A constant number of Protein A/Strep-MB in each assay and a concentration series of the fluorescein-labeled aptamer were used. The dissociation constant (K _D) of 1.06 ±0.2 μM was calculated by nonlinear regression analysis.

Fig 5. Full-length aptamer PA#2/8 and truncated aptamer variants.

The primer binding sites at the 5’- and 3’-end are colored in red and blue, respectively. The G-stretches in the internal sequence region are highlighted in grey.

Fig 6. Binding abilities of the truncated aptamer variants in comparison to the full-length aptamer PA#2/8 to Protein A.

Bead-based binding assays were performed using Protein A/Strep-MB and fluorescein-labeled ssDNA. Target-bound aptamers were eluted and quantified.

Fig 7. SPR interaction analyses concerning the immobilization orientation of aptamer PA#2/8.

Biacore X100 / sensor chip CAP / ligand: 5’-biotinylated aptamer PA#2/8 (A) or 3'-biotinylated aptamer PA#2/8 (B) / analyte: recombinant Protein A with different concentrations (50–2500 nM, 1000 nM in duplicate). Double-referenced sensorgrams are shown (reference surface modified with unselected SELEX library, buffer injection). Black lines represent the fit to bivalent analyte binding model.

Fig 8. SPR interaction analyses regarding the affinity of aptamer PA#2/8.

Biacore X100 / sensor chip CAP / ligand: 3'-biotinylated aptamer PA#2/8 with immobilization levels of 1086 RU (A), 1158 RU (C), 506 RU (E), 316 RU (F) / analyte: recombinant and native Protein A with different concentrations (10–5000 nM, 1000 nM in triplicate) / single-cycle mode (H) with an aptamer level of 516 RU and sequential injections of five ascending concentrations of recombinant Protein A (62, 185, 556, 1667, 5000 nM) as triplicate. Double-referenced sensorgrams are shown (reference surface modified with unselected SELEX library, buffer injection). Black lines represent the fit to bivalent analyte binding model. The corresponding plots (B, D, G, I) of steady-state binding from the end of the association phases against analyte concentration were used to calculate the steady-state affinity.

Fig 9. SPR interaction analysis regarding the affinity of the truncated aptamer PA#2/8[S1-58].

Biacore X100 / sensor chip CAP / ligand: 3'-biotinylated truncated aptamer PA#2/8[S1-58] with immobilization level of 1030 RU / analyte: recombinant Protein A with different concentrations (50–2500 nM, 1000 nM in duplicate). Double-referenced sensorgram (A) is shown (reference surface modified with unselected SELEX library, buffer injection). Black lines represent the fit to bivalent analyte binding model. The corresponding plot (B) of steady-state binding from the end of the association phases against analyte concentration is used to calculate the steady-state affinity.

Fig 10. SPR interaction analyses applying the aptamers PA#2/8 and PA#2/8[S1-58] as analyte.

Biacore X100 / sensor chip CAP / ligand: biotinylated native Protein A with immobilization level of ~600 RU / analyte: 5’-fluorescein-labeled aptamer PA#2/8 (A) or truncated variant PA#2/8[S1-58] (C) with different concentrations (250–8000 nM, 2000 nM in triplicate). Double-referenced sensorgrams are shown (blank reference surface without Protein A, buffer injection). Black lines represent the fit to 1:1 binding model. The corresponding plots (B, D) of steady-state binding from the end of the association phases against analyte concentration are used to calculate the steady-state affinity.

Fig 11. SPR interaction analyses regarding the specificity of aptamer PA#2/8.

Biacore X100 / sensor chip CAP / ligand: 3'-biotinylated aptamer PA#2/8 with immobilization level of 1000–1200 RU / analyte: different proteins with a concentration of 1000 nM each (recombinant Protein A, Protein G, and Protein L in triplicate; HSA, BSA, and human Thrombin in duplicate). Double-referenced sensorgrams (A) are shown (reference surface modified with unselected SELEX library, buffer injection). Bar graph (B) of binding levels of the different proteins from the end of the association phases (after 300 s of injection) is presented.

Fig 12. SPR interaction analyses regarding the aptamer binding site in Protein A.

Biacore X100 / sensor chip CAP / ligand: biotinylated Protein A with immobilization level of ~560 RU / two-step analyte binding without regeneration in between, (A-B) analyte 1 = sample 1: human IgG, IgG-Fc fragment, IgG-Fab fragment with a concentration of 1000 nM each, or buffer, (C-D) analyte 1 = sample 1: concentration series of human IgG-Fc in the range of 0–1000 nM, (A-D) analyte 2 = sample 2: 2000 nM 5’-fluorescein-labeled aptamer PA#2/8 or buffer. Double-referenced sensorgrams are shown (blank reference surface without Protein A, buffer injection). Binding of sample 1 followed by sample 2 is shown in (A) and (C) with alignment to injection start of sample 1. In (B) and (D) only binding of sample 2 with alignment to injection start of sample 2 is shown.

Fig 13. Aptamer—Protein A interactions analyzed by MST.

Binding curves for the interactions of fluorescently labeled aptamer PA#2/8 (A, D) or its truncated variants PA#2/8[S1-58] (B, E) and PA#2/8[S1-43] (C, F) with recombinant or native Protein A are shown. BSA was used as negative control (A). The aptamer concentration was kept constant at 50 nM for each interaction analysis and the protein was titrated in the range from 1.69 to 55,555 nM of recombinant Protein A and from 0.33 to 10,820 nM of native Protein A, respectively. The binding data were fitted, and the dissociation constants (K _D) were calculated.

Fig 14. Overview of calculated dissociation constants (K _D) of aptamer PA#2/8 and its truncated variants.

Results of the applied assays with different measuring principles were compared.