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Aptamer selection based on G4-forming promoter region.

Yoshida W, Saito T, Yokoyama T, Ferri S, Ikebukuro K - PLoS ONE (2013)

Bottom Line: We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products.We designated this aptamer identification method as "G4 promoter-derived aptamer selection (G4PAS)." Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers.In the human genome, over 40% of promoters contain one or more potential G4 DNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture & Technology, Koganei, Tokyo, Japan.

ABSTRACT
We developed a method for aptamer identification without in vitro selection. We have previously obtained several aptamers, which may fold into the G-quadruplex (G4) structure, against target proteins; therefore, we hypothesized that the G4 structure would be an excellent scaffold for aptamers to recognize the target protein. Moreover, the G4-forming sequence contained in the promoter region of insulin can reportedly bind to insulin. We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products. We designated this aptamer identification method as "G4 promoter-derived aptamer selection (G4PAS)." Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers. Surface plasmon resonance (SPR) analysis revealed that the dissociation constant (K d) values of VEGF165, PDGF-AA, and RB1 DNA aptamers were 1.7 × 10(-7) M, 6.3 × 10(-9) M, and 4.4 × 10(-7) M, respectively. G4PAS is a simple and rapid method of aptamer identification because it involves only binding analysis of G4 DNAs to the target protein. In the human genome, over 40% of promoters contain one or more potential G4 DNAs. G4PAS could therefore be applied to identify aptamers against target proteins that contain G4 DNAs on their promoters.

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Gel shift competition assay.In the presence of FITC-labeled VEGFA G4 (0.5 µM), non-labeled PGDFA G4 (0.5 µM) or RB1 G4 (0.5 µM) was incubated with VEGF165 (2.6 µM). The mixtures were electrophoresed on 12% polyacrylamide gel in TBE buffer, and FITC images were then detected.
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pone-0065497-g004: Gel shift competition assay.In the presence of FITC-labeled VEGFA G4 (0.5 µM), non-labeled PGDFA G4 (0.5 µM) or RB1 G4 (0.5 µM) was incubated with VEGF165 (2.6 µM). The mixtures were electrophoresed on 12% polyacrylamide gel in TBE buffer, and FITC images were then detected.

Mentions: We also performed a gel shift competition assay to investigate whether VEGFA G4, PDGFA G4, and RB1 G4 bind to the same site of VEGF165. In the presence or absence of VEGF165, equal moles of 5′-FITC-labeled VEGFA G4 and non-labeled PDGFA G4 or RB1 G4 were mixed and then analyzed by native PAGE. We did not detect a band shift of VEGFA G4 in the presence of PDGFA G4 (Figure 4). These results suggest that VEGFA G4 and PDGFA G4 recognize the same site on the heparin-binding domain of VEGF165. However, we detected a band shift of VEGFA G4 in the presence of RB1 G4. This result suggests that RB1 G4 would recognize a different site on the heparin-binding domain or that competitive binding was not observed because of the lower binding affinity of RB1 G4 to VEGF165.


Aptamer selection based on G4-forming promoter region.

Yoshida W, Saito T, Yokoyama T, Ferri S, Ikebukuro K - PLoS ONE (2013)

Gel shift competition assay.In the presence of FITC-labeled VEGFA G4 (0.5 µM), non-labeled PGDFA G4 (0.5 µM) or RB1 G4 (0.5 µM) was incubated with VEGF165 (2.6 µM). The mixtures were electrophoresed on 12% polyacrylamide gel in TBE buffer, and FITC images were then detected.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672139&req=5

pone-0065497-g004: Gel shift competition assay.In the presence of FITC-labeled VEGFA G4 (0.5 µM), non-labeled PGDFA G4 (0.5 µM) or RB1 G4 (0.5 µM) was incubated with VEGF165 (2.6 µM). The mixtures were electrophoresed on 12% polyacrylamide gel in TBE buffer, and FITC images were then detected.
Mentions: We also performed a gel shift competition assay to investigate whether VEGFA G4, PDGFA G4, and RB1 G4 bind to the same site of VEGF165. In the presence or absence of VEGF165, equal moles of 5′-FITC-labeled VEGFA G4 and non-labeled PDGFA G4 or RB1 G4 were mixed and then analyzed by native PAGE. We did not detect a band shift of VEGFA G4 in the presence of PDGFA G4 (Figure 4). These results suggest that VEGFA G4 and PDGFA G4 recognize the same site on the heparin-binding domain of VEGF165. However, we detected a band shift of VEGFA G4 in the presence of RB1 G4. This result suggests that RB1 G4 would recognize a different site on the heparin-binding domain or that competitive binding was not observed because of the lower binding affinity of RB1 G4 to VEGF165.

Bottom Line: We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products.We designated this aptamer identification method as "G4 promoter-derived aptamer selection (G4PAS)." Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers.In the human genome, over 40% of promoters contain one or more potential G4 DNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture & Technology, Koganei, Tokyo, Japan.

ABSTRACT
We developed a method for aptamer identification without in vitro selection. We have previously obtained several aptamers, which may fold into the G-quadruplex (G4) structure, against target proteins; therefore, we hypothesized that the G4 structure would be an excellent scaffold for aptamers to recognize the target protein. Moreover, the G4-forming sequence contained in the promoter region of insulin can reportedly bind to insulin. We thus expected that G4 DNAs, which are contained in promoter regions, could act as DNA aptamers against their gene products. We designated this aptamer identification method as "G4 promoter-derived aptamer selection (G4PAS)." Using G4PAS, we identified vascular endothelial growth factor (VEGF)165, platelet-derived growth factor-AA (PDGF)-AA, and RB1 DNA aptamers. Surface plasmon resonance (SPR) analysis revealed that the dissociation constant (K d) values of VEGF165, PDGF-AA, and RB1 DNA aptamers were 1.7 × 10(-7) M, 6.3 × 10(-9) M, and 4.4 × 10(-7) M, respectively. G4PAS is a simple and rapid method of aptamer identification because it involves only binding analysis of G4 DNAs to the target protein. In the human genome, over 40% of promoters contain one or more potential G4 DNAs. G4PAS could therefore be applied to identify aptamers against target proteins that contain G4 DNAs on their promoters.

Show MeSH
Related in: MedlinePlus