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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 binding assay of G4-forming DNAs to target proteins.In the presence or absence of targets, fluorescent-labeled G4-forming DNAs were electrophoresed on 12% polyacrylamide gel in TBE buffer, and fluorescence images were then detected. Arrows indicate bands of DNA–protein complex. Gel shift assay of VEGFA G4 to VEGF165 (a), PDGFA G4 to PDGF-AA (b), RB1 G4 to RB1 (c), c-KIT G4-1 to c-KIT (d), and c-KIT G4-2 to c-KIT (e). In the assay of c-KIT G4-1 and c-KIT G4-2, both the intracellular (second lane) and extracellular (third lane) domains of c-KIT were used.
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pone-0065497-g001: Gel shift binding assay of G4-forming DNAs to target proteins.In the presence or absence of targets, fluorescent-labeled G4-forming DNAs were electrophoresed on 12% polyacrylamide gel in TBE buffer, and fluorescence images were then detected. Arrows indicate bands of DNA–protein complex. Gel shift assay of VEGFA G4 to VEGF165 (a), PDGFA G4 to PDGF-AA (b), RB1 G4 to RB1 (c), c-KIT G4-1 to c-KIT (d), and c-KIT G4-2 to c-KIT (e). In the assay of c-KIT G4-1 and c-KIT G4-2, both the intracellular (second lane) and extracellular (third lane) domains of c-KIT were used.

Mentions: Several promoter regions reportedly contain G4-forming DNA. To investigate whether the G4-forming DNAs bind to their protein products in vitro, we focused on the VEGFA, PDGFA, RB1, and c-KIT genes. The VEGFA promoter contains one parallel G4 structure located −50 to −85 bp upstream of the transcription start site (TSS), the PDGFA promoter contains one parallel G4 structure located −47 to −82 bp upstream of TSS, the RB1 gene contains one antiparallel G4 structure located +169 to +186 bp downstream of TSS, and the c-KIT promoter contains two G4 structures located −74 to −94 bp, and −23 to −44 bp upstream of TSS. We designated these G4 DNAs as VEGFA G4, PDGFA G4, RB1 G4, and c-KIT G4-1 and c-KIT G4-2, respectively (Table 1). To perform the gel shift assay, these fluorescent-labeled G4 DNAs were synthesized. We confirmed that these fluorescence-labeled DNAs form a parallel G-quadruplex structure, which is similar to that formed by non-labeled DNAs, as determined by measurement of CD spectra (Figure S1). We used the primary isoform of VEGFA (VEGF165) and PDGF-AA as the target proteins of VEGFA G4 and PDGFA G4, respectively. We also used the intracellular and extracellular domains of c-KIT proteins as the target proteins of c-KIT G4 because c-KIT is a membrane protein. In the gel shift assay, we observed a band shift of VEGFA G4, PDGFA G4, and RB1 G4 but not of c-KIT G4s (Figure 1). At the position of the shifted band of these oligonucleotides, we also detected these proteins by silver staining. These results indicated that VEGFA G4, PDGFA G4, and RB1 G4 bound to VEGF165, PDGF-AA, and RB1 protein in vitro, respectively; however, c-KIT G4-1 and G4-2 DNAs did not bind to the intracellular and extracellular domains of c-KIT protein. We observed both monomeric and multimeric PDGFA G4 in the absence of PDGF-AA; however, the band of monomeric PDGFA G4 was completely shifted in the presence of PDGF-AA, suggesting that monomeric PDGFA G4 would bind to PDGF-AA.


Aptamer selection based on G4-forming promoter region.

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

Gel shift binding assay of G4-forming DNAs to target proteins.In the presence or absence of targets, fluorescent-labeled G4-forming DNAs were electrophoresed on 12% polyacrylamide gel in TBE buffer, and fluorescence images were then detected. Arrows indicate bands of DNA–protein complex. Gel shift assay of VEGFA G4 to VEGF165 (a), PDGFA G4 to PDGF-AA (b), RB1 G4 to RB1 (c), c-KIT G4-1 to c-KIT (d), and c-KIT G4-2 to c-KIT (e). In the assay of c-KIT G4-1 and c-KIT G4-2, both the intracellular (second lane) and extracellular (third lane) domains of c-KIT were used.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672139&req=5

pone-0065497-g001: Gel shift binding assay of G4-forming DNAs to target proteins.In the presence or absence of targets, fluorescent-labeled G4-forming DNAs were electrophoresed on 12% polyacrylamide gel in TBE buffer, and fluorescence images were then detected. Arrows indicate bands of DNA–protein complex. Gel shift assay of VEGFA G4 to VEGF165 (a), PDGFA G4 to PDGF-AA (b), RB1 G4 to RB1 (c), c-KIT G4-1 to c-KIT (d), and c-KIT G4-2 to c-KIT (e). In the assay of c-KIT G4-1 and c-KIT G4-2, both the intracellular (second lane) and extracellular (third lane) domains of c-KIT were used.
Mentions: Several promoter regions reportedly contain G4-forming DNA. To investigate whether the G4-forming DNAs bind to their protein products in vitro, we focused on the VEGFA, PDGFA, RB1, and c-KIT genes. The VEGFA promoter contains one parallel G4 structure located −50 to −85 bp upstream of the transcription start site (TSS), the PDGFA promoter contains one parallel G4 structure located −47 to −82 bp upstream of TSS, the RB1 gene contains one antiparallel G4 structure located +169 to +186 bp downstream of TSS, and the c-KIT promoter contains two G4 structures located −74 to −94 bp, and −23 to −44 bp upstream of TSS. We designated these G4 DNAs as VEGFA G4, PDGFA G4, RB1 G4, and c-KIT G4-1 and c-KIT G4-2, respectively (Table 1). To perform the gel shift assay, these fluorescent-labeled G4 DNAs were synthesized. We confirmed that these fluorescence-labeled DNAs form a parallel G-quadruplex structure, which is similar to that formed by non-labeled DNAs, as determined by measurement of CD spectra (Figure S1). We used the primary isoform of VEGFA (VEGF165) and PDGF-AA as the target proteins of VEGFA G4 and PDGFA G4, respectively. We also used the intracellular and extracellular domains of c-KIT proteins as the target proteins of c-KIT G4 because c-KIT is a membrane protein. In the gel shift assay, we observed a band shift of VEGFA G4, PDGFA G4, and RB1 G4 but not of c-KIT G4s (Figure 1). At the position of the shifted band of these oligonucleotides, we also detected these proteins by silver staining. These results indicated that VEGFA G4, PDGFA G4, and RB1 G4 bound to VEGF165, PDGF-AA, and RB1 protein in vitro, respectively; however, c-KIT G4-1 and G4-2 DNAs did not bind to the intracellular and extracellular domains of c-KIT protein. We observed both monomeric and multimeric PDGFA G4 in the absence of PDGF-AA; however, the band of monomeric PDGFA G4 was completely shifted in the presence of PDGF-AA, suggesting that monomeric PDGFA G4 would bind to PDGF-AA.

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