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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

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 (KD) of 1.06 ±0.2 μM was calculated by nonlinear regression analysis.
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pone.0134403.g004: 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 (KD) of 1.06 ±0.2 μM was calculated by nonlinear regression analysis.

Mentions: Bead-based binding assays according to the selection conditions revealed a concentration dependent binding of aptamer PA#2/8 to Protein A/Strep-MB (Fig 4). A concentration range of 12 nM—3600 nM fluorescein-labeled aptamer was applied to a series of individual binding assays with a constant number of target-modified beads. On the basis of these binding data, a saturation curve was obtained and a dissociation constant of KD = 1.06 ±0.2 μM was calculated.


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

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

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 (KD) of 1.06 ±0.2 μM was calculated by nonlinear regression analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519192&req=5

pone.0134403.g004: 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 (KD) of 1.06 ±0.2 μM was calculated by nonlinear regression analysis.
Mentions: Bead-based binding assays according to the selection conditions revealed a concentration dependent binding of aptamer PA#2/8 to Protein A/Strep-MB (Fig 4). A concentration range of 12 nM—3600 nM fluorescein-labeled aptamer was applied to a series of individual binding assays with a constant number of target-modified beads. On the basis of these binding data, a saturation curve was obtained and a dissociation constant of KD = 1.06 ±0.2 μM was calculated.

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