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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 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.
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pone.0134403.g008: 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.

Mentions: In addition to the bead-based binding assays, SPR measurements were used to analyze the binding affinity of aptamer PA#2/8 to its target Protein A in more detail. In the first experimental setup, the sensor surface was build up by immobilization of 3’-biotinylated aptamer on the streptavidin-coated sensor chip, and a concentration series of Protein A (native or recombinant) in the range of 10–5000 nM was injected. An aptamer level of 1000–1200 RU on the sensor surface was adjusted in each experiment over the injection time during the immobilization of the aptamer. The sensorgrams in Fig 8A and 8C reveal a very tightly and stable binding behavior of both recombinant and native Protein A to the immobilized aptamer PA#2/8. Fig 8B and 8D show the corresponding saturation curves derived from the binding data at the end of the binding phases. Based on this, dissociation constants in the low nanomolar range were calculated: KD = 172 ±14 nM for the recombinant Protein A and KD = 84 ±5 nM for the native 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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134403.g008: 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.
Mentions: In addition to the bead-based binding assays, SPR measurements were used to analyze the binding affinity of aptamer PA#2/8 to its target Protein A in more detail. In the first experimental setup, the sensor surface was build up by immobilization of 3’-biotinylated aptamer on the streptavidin-coated sensor chip, and a concentration series of Protein A (native or recombinant) in the range of 10–5000 nM was injected. An aptamer level of 1000–1200 RU on the sensor surface was adjusted in each experiment over the injection time during the immobilization of the aptamer. The sensorgrams in Fig 8A and 8C reveal a very tightly and stable binding behavior of both recombinant and native Protein A to the immobilized aptamer PA#2/8. Fig 8B and 8D show the corresponding saturation curves derived from the binding data at the end of the binding phases. Based on this, dissociation constants in the low nanomolar range were calculated: KD = 172 ±14 nM for the recombinant Protein A and KD = 84 ±5 nM for the native 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