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

Full-length aptamer PA#2/8 and truncated aptamer variants.The primer binding sites at the 5’- and 3’-end are colored in red and blue, respectively. The G-stretches in the internal sequence region are highlighted in grey.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134403.g005: Full-length aptamer PA#2/8 and truncated aptamer variants.The primer binding sites at the 5’- and 3’-end are colored in red and blue, respectively. The G-stretches in the internal sequence region are highlighted in grey.

Mentions: The aptamer PA#2/8 was stepwise truncated to narrow down the aptamer sequence region responsible for target binding. All of the truncated aptamer variants were fluorescein-labeled at the 5’-end and tested for their functionality using bead-based binding assays with Protein A/Strep-MB. Firstly either one of the specific primer binding sites at the ends of aptamer PA#2/8 (PA#2/8[S19-76], PA#2/8[S1-58]) or both of them (PA#2/8KR40, PA#2/8KR23) were removed (Fig 5). Only aptamer variant PA#2/8[S1-58] was able to continue to bind to Protein A even with a higher binding signal than the full-length aptamer (Fig 6). No binding signals were observed for the variants PA#2/8[S19-76], PA#2/8KR40 and PA#2/8KR23. These results clearly indicate that the 5’-primer binding site of PA#2/8 is very important for the functionality of the aptamer and seems to be involved in its functional folding. A loss of the 18 nt at the 5’-end of aptamer PA#2/8 leads to a completely loss of its binding ability to Protein A. In contrast, the 18 nt at the 3’-end of PA#2/8 are not necessary for target binding.


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

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

Full-length aptamer PA#2/8 and truncated aptamer variants.The primer binding sites at the 5’- and 3’-end are colored in red and blue, respectively. The G-stretches in the internal sequence region are highlighted in grey.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134403.g005: Full-length aptamer PA#2/8 and truncated aptamer variants.The primer binding sites at the 5’- and 3’-end are colored in red and blue, respectively. The G-stretches in the internal sequence region are highlighted in grey.
Mentions: The aptamer PA#2/8 was stepwise truncated to narrow down the aptamer sequence region responsible for target binding. All of the truncated aptamer variants were fluorescein-labeled at the 5’-end and tested for their functionality using bead-based binding assays with Protein A/Strep-MB. Firstly either one of the specific primer binding sites at the ends of aptamer PA#2/8 (PA#2/8[S19-76], PA#2/8[S1-58]) or both of them (PA#2/8KR40, PA#2/8KR23) were removed (Fig 5). Only aptamer variant PA#2/8[S1-58] was able to continue to bind to Protein A even with a higher binding signal than the full-length aptamer (Fig 6). No binding signals were observed for the variants PA#2/8[S19-76], PA#2/8KR40 and PA#2/8KR23. These results clearly indicate that the 5’-primer binding site of PA#2/8 is very important for the functionality of the aptamer and seems to be involved in its functional folding. A loss of the 18 nt at the 5’-end of aptamer PA#2/8 leads to a completely loss of its binding ability to Protein A. In contrast, the 18 nt at the 3’-end of PA#2/8 are not necessary for target binding.

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