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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 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.
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pone.0134403.g007: 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.

Mentions: Surface plasmon resonance (SPR) based technologies stand for label-free detection methods and allow very efficient interaction analyses between aptamers and their targets on a surface. In this work, the Biacore X100 instrument was used to characterize the binding features of aptamer PA#2/8 to Protein A. In most of the experiments, end-labeled aptamer with biotin was immobilized on the streptavidin-modified sensor surface of sensor chip CAP of the Biotin CAPture Kit, and Protein A was injected as analyte across this surface for interaction with the aptamer. The first experiments have clearly shown that in case of aptamer PA#2/8 the immobilization site of this aptamer is very important for the maintenance of its functionality to bind Protein A. Immobilization of aptamer PA#2/8 at its 5’-end resulted in a very weak binding of Protein A to the aptamer layer on the sensor surface (Fig 7). In contrast, a very good and concentration-dependent binding of Protein A could be observed using a sensor surface with 3’-immobilized aptamer. This indicates that a free 5'- end of the aptamer is necessary for its correct folding as basis for formation of the binding complex with the target. These findings are in accordance with the results of the truncation experiments showing that an intact 5’-primer binding site of the aptamer must be involved in the functional folding for binding to 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 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519192&req=5

pone.0134403.g007: 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.
Mentions: Surface plasmon resonance (SPR) based technologies stand for label-free detection methods and allow very efficient interaction analyses between aptamers and their targets on a surface. In this work, the Biacore X100 instrument was used to characterize the binding features of aptamer PA#2/8 to Protein A. In most of the experiments, end-labeled aptamer with biotin was immobilized on the streptavidin-modified sensor surface of sensor chip CAP of the Biotin CAPture Kit, and Protein A was injected as analyte across this surface for interaction with the aptamer. The first experiments have clearly shown that in case of aptamer PA#2/8 the immobilization site of this aptamer is very important for the maintenance of its functionality to bind Protein A. Immobilization of aptamer PA#2/8 at its 5’-end resulted in a very weak binding of Protein A to the aptamer layer on the sensor surface (Fig 7). In contrast, a very good and concentration-dependent binding of Protein A could be observed using a sensor surface with 3’-immobilized aptamer. This indicates that a free 5'- end of the aptamer is necessary for its correct folding as basis for formation of the binding complex with the target. These findings are in accordance with the results of the truncation experiments showing that an intact 5’-primer binding site of the aptamer must be involved in the functional folding for binding to 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