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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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SPR analysis of binding of G4-forming DNAs to target proteins.(a) SPR binding signal of VEGFA G4 (top) and VEGFA G4 mutant (bottom) to VEGF165 immobilized on a CM5 chip. (b) SPR binding signal of PDGFA G4 (top) or PDGFA G4 mutant (bottom) to PDGF-AA immobilized on a CM5 chip. (c) SPR binding signal of RB1 to RB1 G4 immobilized on an SA chip (top) or RB1 G4 mutant immobilized on an SA chip (bottom).
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pone-0065497-g002: SPR analysis of binding of G4-forming DNAs to target proteins.(a) SPR binding signal of VEGFA G4 (top) and VEGFA G4 mutant (bottom) to VEGF165 immobilized on a CM5 chip. (b) SPR binding signal of PDGFA G4 (top) or PDGFA G4 mutant (bottom) to PDGF-AA immobilized on a CM5 chip. (c) SPR binding signal of RB1 to RB1 G4 immobilized on an SA chip (top) or RB1 G4 mutant immobilized on an SA chip (bottom).

Mentions: We next investigated the binding kinetics of VEGFA G4, PDGFA G4, and RB1 G4 to their target proteins by SPR. In SPR analysis of VEGFA G4 and PDGFA G4, the target proteins were immobilized on a CM5 chip via amine coupling and non-labeled oligonucleotides were then applied to the sensor chip. In SPR analysis of RB1 G4, biotinylated oligonucleotides were immobilized on the sensor chip SA and RB1 protein was then applied to the sensor chip. As a control, we used mutant VEGFA G4, PDGFA G4, and RB1 G4 that were not expected to form the G4 structure (Table 1). In SPR analysis, we detected the binding signal of VEGFA G4, PDGFA G4, and RB1 G4 to the target proteins, and their Kd values were calculated to be 1.7×10−7 M, 6.3×10−9 M, and 4.4×10−7 M, respectively (Figure 2). The Kd value of VEGFA G4 to VEGF165 was similar to that of the VEGF165 DNA aptamer obtained by SELEX [40], and PDGFA G4 bound to PDGF-AA at a nanomolar level, suggesting that DNA aptamers that bind to the target protein with high affinity can be obtained by G4PAS. However, mutant VEGFA G4, PDGFA G4, and RB1 G4 did not bind to their target proteins. These results indicated that the G4 structures are important for recognizing their target proteins.


Aptamer selection based on G4-forming promoter region.

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

SPR analysis of binding of G4-forming DNAs to target proteins.(a) SPR binding signal of VEGFA G4 (top) and VEGFA G4 mutant (bottom) to VEGF165 immobilized on a CM5 chip. (b) SPR binding signal of PDGFA G4 (top) or PDGFA G4 mutant (bottom) to PDGF-AA immobilized on a CM5 chip. (c) SPR binding signal of RB1 to RB1 G4 immobilized on an SA chip (top) or RB1 G4 mutant immobilized on an SA chip (bottom).
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pone-0065497-g002: SPR analysis of binding of G4-forming DNAs to target proteins.(a) SPR binding signal of VEGFA G4 (top) and VEGFA G4 mutant (bottom) to VEGF165 immobilized on a CM5 chip. (b) SPR binding signal of PDGFA G4 (top) or PDGFA G4 mutant (bottom) to PDGF-AA immobilized on a CM5 chip. (c) SPR binding signal of RB1 to RB1 G4 immobilized on an SA chip (top) or RB1 G4 mutant immobilized on an SA chip (bottom).
Mentions: We next investigated the binding kinetics of VEGFA G4, PDGFA G4, and RB1 G4 to their target proteins by SPR. In SPR analysis of VEGFA G4 and PDGFA G4, the target proteins were immobilized on a CM5 chip via amine coupling and non-labeled oligonucleotides were then applied to the sensor chip. In SPR analysis of RB1 G4, biotinylated oligonucleotides were immobilized on the sensor chip SA and RB1 protein was then applied to the sensor chip. As a control, we used mutant VEGFA G4, PDGFA G4, and RB1 G4 that were not expected to form the G4 structure (Table 1). In SPR analysis, we detected the binding signal of VEGFA G4, PDGFA G4, and RB1 G4 to the target proteins, and their Kd values were calculated to be 1.7×10−7 M, 6.3×10−9 M, and 4.4×10−7 M, respectively (Figure 2). The Kd value of VEGFA G4 to VEGF165 was similar to that of the VEGF165 DNA aptamer obtained by SELEX [40], and PDGFA G4 bound to PDGF-AA at a nanomolar level, suggesting that DNA aptamers that bind to the target protein with high affinity can be obtained by G4PAS. However, mutant VEGFA G4, PDGFA G4, and RB1 G4 did not bind to their target proteins. These results indicated that the G4 structures are important for recognizing their target proteins.

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