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

Stoltenburg R, Schubert T, Strehlitz B - PLoS ONE (2015)

Bottom Line: 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.Cross specificity to other proteins was not found.The application of the aptamer is directed to Protein A detection or affinity purification.

View Article: PubMed Central - PubMed

Affiliation: UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0134403.g012: 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.

Mentions: Two-step analyte binding experiments without regeneration in between were performed using the Biacore X100 instrument to prove the hypothesis that aptamer PA#2/8 interacts with Protein A at the same sites as immunoglobulins. For this purpose, biotinylated Protein A was immobilized on the sensor surface (~560 RU). Different cycles were run using human IgG, IgG-Fc fragment, IgG-Fab fragment (1000 nM each), or only buffer for the first sample injection to allow binding to Protein A followed by dissociation (Fig 12A). Afterwards aptamer PA#2/8 (2000 nM) or only buffer representing the second sample was directly injected for binding. Not until after the second dissociation phase the sensor surface was regenerated for a new cycle. As expected, human IgG and its Fc fragment are able to bind very tightly and stable to Protein A on the sensor surface (Fig 12A), and resulted in a very strong signal. A relatively weak binding signal was observed for the human Fab fragment. Less common is the ability of Protein A to bind the Fab fragment of immunoglobulins, especially the Fab heavy chain VH3 family [7, 8, 43, 44]. It was found that both Fc and Fab fragments bind to Protein A in a noncompetitive way and that they use the same Ig-binding domains, but different epitopes inside of these domains [44]. If IgG or the IgG-Fc fragment was bound to Protein A first, a subsequent interaction of aptamer PA#2/8 with the same sensor surface could not be found (Fig 12B). A typical binding signal for the aptamer was only observed after using buffer or the Fab fragment as analyte during the first binding step. Fig 12C and 12D show a similar experiment with the injection of a concentration series of IgG-Fc in the range of 0–1000 nM during the first binding step. In this case, the aptamer PA#2/8 was able to bind to Protein A afterwards in dependence of the amount of bound IgG-Fc on the sensor surface. From the results of both experiments, it can be concluded that aptamer PA#2/8 compete with IgG or IgG-Fc for binding to Protein A and therefore use the same sites as immunoglobulins for interaction with Protein A.


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

Stoltenburg R, Schubert T, Strehlitz B - PLoS ONE (2015)

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.
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Related In: Results  -  Collection

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pone.0134403.g012: 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.
Mentions: Two-step analyte binding experiments without regeneration in between were performed using the Biacore X100 instrument to prove the hypothesis that aptamer PA#2/8 interacts with Protein A at the same sites as immunoglobulins. For this purpose, biotinylated Protein A was immobilized on the sensor surface (~560 RU). Different cycles were run using human IgG, IgG-Fc fragment, IgG-Fab fragment (1000 nM each), or only buffer for the first sample injection to allow binding to Protein A followed by dissociation (Fig 12A). Afterwards aptamer PA#2/8 (2000 nM) or only buffer representing the second sample was directly injected for binding. Not until after the second dissociation phase the sensor surface was regenerated for a new cycle. As expected, human IgG and its Fc fragment are able to bind very tightly and stable to Protein A on the sensor surface (Fig 12A), and resulted in a very strong signal. A relatively weak binding signal was observed for the human Fab fragment. Less common is the ability of Protein A to bind the Fab fragment of immunoglobulins, especially the Fab heavy chain VH3 family [7, 8, 43, 44]. It was found that both Fc and Fab fragments bind to Protein A in a noncompetitive way and that they use the same Ig-binding domains, but different epitopes inside of these domains [44]. If IgG or the IgG-Fc fragment was bound to Protein A first, a subsequent interaction of aptamer PA#2/8 with the same sensor surface could not be found (Fig 12B). A typical binding signal for the aptamer was only observed after using buffer or the Fab fragment as analyte during the first binding step. Fig 12C and 12D show a similar experiment with the injection of a concentration series of IgG-Fc in the range of 0–1000 nM during the first binding step. In this case, the aptamer PA#2/8 was able to bind to Protein A afterwards in dependence of the amount of bound IgG-Fc on the sensor surface. From the results of both experiments, it can be concluded that aptamer PA#2/8 compete with IgG or IgG-Fc for binding to Protein A and therefore use the same sites as immunoglobulins for interaction with Protein A.

Bottom Line: 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.Cross specificity to other proteins was not found.The application of the aptamer is directed to Protein A detection or affinity purification.

View Article: PubMed Central - PubMed

Affiliation: UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle, Germany.

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.

No MeSH data available.


Related in: MedlinePlus