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SDA, a DNA aptamer inhibiting E- and P-selectin mediated adhesion of cancer and leukemia cells, the first and pivotal step in transendothelial migration during metastasis formation.

Faryammanesh R, Lange T, Magbanua E, Haas S, Meyer C, Wicklein D, Schumacher U, Hahn U - PLoS ONE (2014)

Bottom Line: We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K(d) value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands.Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro.In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.

View Article: PubMed Central - PubMed

Affiliation: Hamburg University, MIN-Faculty, Chemistry Department, Institute for Biochemistry and Molecular Biology, Hamburg, Germany.

ABSTRACT
Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation in vivo. We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K(d) value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.

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Related in: MedlinePlus

Systematic Evolution of Ligands by Exponential Enrichment (SELEX).DNA was incubated with immobilized rh E-selectin on magnetic beads (target beads). After washing and removing unbound DNA, target bound DNA was eluted and amplified via PCR. The double-stranded PCR product was separated in single strand DNA and the next SELEX step started after this regeneration. The identification of aptamers via cloning and sequencing of the enriched ssDNA pool was performed after several SELEX rounds (10–20 rounds).
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pone-0093173-g002: Systematic Evolution of Ligands by Exponential Enrichment (SELEX).DNA was incubated with immobilized rh E-selectin on magnetic beads (target beads). After washing and removing unbound DNA, target bound DNA was eluted and amplified via PCR. The double-stranded PCR product was separated in single strand DNA and the next SELEX step started after this regeneration. The identification of aptamers via cloning and sequencing of the enriched ssDNA pool was performed after several SELEX rounds (10–20 rounds).

Mentions: For in vitro selection of E-selectin specific DNA aptamers, we used the SELEX procedure (Figure 2) including a pre-selection step to remove streptavidin-binding DNA. For pre-selection, 250 pmol of the ssDNA library (5′-GCCTGTTGTGAGCCTCCTAAC-(N)50-CATGCTTATTCTTGTCTCCC-3′) were incubated with 30 nmol pre-selection beads for 30 minutes at room temperature. Streptavidin-binding DNA was removed via magnetic separation. The first round of selection was initiated by incubation of pre-selected DNA library with 50 pmol target beads for 30 minutes at room temperature. After removing of unbound DNA by washing with 200 μL selection buffer, bound DNA was eluted in 55 μL water by heating the mixture to 80°C for 3 minutes. Eluted DNA was amplified via PCR using 1 μM 5′-biotinylated reverse primer (5′-GGGAGACAAGAATAAGCATG-3′), 1 μM forward primer (5′-GCCTGTTGTGfAGCCTCCTAAC-3′), 1.5 mM MgCl2, 200 μM dNTPs in 1×PCR buffer B and 0.05 U FIREPol DNA Polymerase per μL (the latter two purchased from Solis BioDyne). Separation of single-stranded DNA aptamers from double-stranded PCR products was done by streptavidin-coated magnetic beads. Therefore, PCR products were diluted 1∶2 in 2×B&W buffer (1 mM EDTA, 2 M NaCl in 10 mM Tris-HCl, pH 7.5), added to streptavidin-coated magnetic beads and subsequently washed twice with 1×B&W buffer. After incubation for 15 min at RT the supernatants were removed and beads were re-suspended in 150 mM NaOH following incubation for another 10 minutes at room temperature. The combined supernatants, containing the single-stranded DNA, were neutralized using 100 mM HCl and utilized as start pool for a next selection round. After incubation of DNA single strands with target beads the amounts of washing steps were raised from round to round increasing stringency.


SDA, a DNA aptamer inhibiting E- and P-selectin mediated adhesion of cancer and leukemia cells, the first and pivotal step in transendothelial migration during metastasis formation.

Faryammanesh R, Lange T, Magbanua E, Haas S, Meyer C, Wicklein D, Schumacher U, Hahn U - PLoS ONE (2014)

Systematic Evolution of Ligands by Exponential Enrichment (SELEX).DNA was incubated with immobilized rh E-selectin on magnetic beads (target beads). After washing and removing unbound DNA, target bound DNA was eluted and amplified via PCR. The double-stranded PCR product was separated in single strand DNA and the next SELEX step started after this regeneration. The identification of aptamers via cloning and sequencing of the enriched ssDNA pool was performed after several SELEX rounds (10–20 rounds).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3974700&req=5

pone-0093173-g002: Systematic Evolution of Ligands by Exponential Enrichment (SELEX).DNA was incubated with immobilized rh E-selectin on magnetic beads (target beads). After washing and removing unbound DNA, target bound DNA was eluted and amplified via PCR. The double-stranded PCR product was separated in single strand DNA and the next SELEX step started after this regeneration. The identification of aptamers via cloning and sequencing of the enriched ssDNA pool was performed after several SELEX rounds (10–20 rounds).
Mentions: For in vitro selection of E-selectin specific DNA aptamers, we used the SELEX procedure (Figure 2) including a pre-selection step to remove streptavidin-binding DNA. For pre-selection, 250 pmol of the ssDNA library (5′-GCCTGTTGTGAGCCTCCTAAC-(N)50-CATGCTTATTCTTGTCTCCC-3′) were incubated with 30 nmol pre-selection beads for 30 minutes at room temperature. Streptavidin-binding DNA was removed via magnetic separation. The first round of selection was initiated by incubation of pre-selected DNA library with 50 pmol target beads for 30 minutes at room temperature. After removing of unbound DNA by washing with 200 μL selection buffer, bound DNA was eluted in 55 μL water by heating the mixture to 80°C for 3 minutes. Eluted DNA was amplified via PCR using 1 μM 5′-biotinylated reverse primer (5′-GGGAGACAAGAATAAGCATG-3′), 1 μM forward primer (5′-GCCTGTTGTGfAGCCTCCTAAC-3′), 1.5 mM MgCl2, 200 μM dNTPs in 1×PCR buffer B and 0.05 U FIREPol DNA Polymerase per μL (the latter two purchased from Solis BioDyne). Separation of single-stranded DNA aptamers from double-stranded PCR products was done by streptavidin-coated magnetic beads. Therefore, PCR products were diluted 1∶2 in 2×B&W buffer (1 mM EDTA, 2 M NaCl in 10 mM Tris-HCl, pH 7.5), added to streptavidin-coated magnetic beads and subsequently washed twice with 1×B&W buffer. After incubation for 15 min at RT the supernatants were removed and beads were re-suspended in 150 mM NaOH following incubation for another 10 minutes at room temperature. The combined supernatants, containing the single-stranded DNA, were neutralized using 100 mM HCl and utilized as start pool for a next selection round. After incubation of DNA single strands with target beads the amounts of washing steps were raised from round to round increasing stringency.

Bottom Line: We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K(d) value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands.Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro.In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.

View Article: PubMed Central - PubMed

Affiliation: Hamburg University, MIN-Faculty, Chemistry Department, Institute for Biochemistry and Molecular Biology, Hamburg, Germany.

ABSTRACT
Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation in vivo. We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K(d) value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.

Show MeSH
Related in: MedlinePlus