Limits...
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 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4519192&req=5

pone.0134403.g011: 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.

Mentions: SPR measurements were also used to characterize the binding features of aptamer PA#2/8 regarding its specificity. An overlay of sensorgrams showing interactions between different proteins and immobilized aptamer is presented in Fig 11A. Aptamer PA#2/8 effectively distinguishes the target Protein A from the functionally related proteins Protein G and Protein L. Both proteins are also immunoglobulin-binding proteins and like Protein A commonly used to purify, immobilize, or detect immunoglobulins [5]. On the other hand, there are strong differences between these three proteins concerning their origins or structural features. Furthermore, each of these proteins has a different immunoglobulin-binding profile, e.g., in terms of the binding site, the species, and type of Ig. Protein G is a bacterial cell wall protein from group G Streptococci [37–39]. It contains two Ig-binding domains as well as sites for albumin and cell surface binding, which were eliminated from the recombinant Protein G expressed in E.coli and used in this work. Protein L is a bacterial cell surface protein from Peptostreptococcus magnus. In contrast to Protein A and Protein G, which bind to the Fc region of immunoglobulins, Protein L binds Igs through light chain interactions, but without interfering with the antigen-binding site, and also binds to single chain variable fragments (scFv) and Fab fragments. The recombinant variant of this protein expressed in E.coli was used in this work [40–42].


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134403.g011: 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.
Mentions: SPR measurements were also used to characterize the binding features of aptamer PA#2/8 regarding its specificity. An overlay of sensorgrams showing interactions between different proteins and immobilized aptamer is presented in Fig 11A. Aptamer PA#2/8 effectively distinguishes the target Protein A from the functionally related proteins Protein G and Protein L. Both proteins are also immunoglobulin-binding proteins and like Protein A commonly used to purify, immobilize, or detect immunoglobulins [5]. On the other hand, there are strong differences between these three proteins concerning their origins or structural features. Furthermore, each of these proteins has a different immunoglobulin-binding profile, e.g., in terms of the binding site, the species, and type of Ig. Protein G is a bacterial cell wall protein from group G Streptococci [37–39]. It contains two Ig-binding domains as well as sites for albumin and cell surface binding, which were eliminated from the recombinant Protein G expressed in E.coli and used in this work. Protein L is a bacterial cell surface protein from Peptostreptococcus magnus. In contrast to Protein A and Protein G, which bind to the Fc region of immunoglobulins, Protein L binds Igs through light chain interactions, but without interfering with the antigen-binding site, and also binds to single chain variable fragments (scFv) and Fab fragments. The recombinant variant of this protein expressed in E.coli was used in this work [40–42].

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