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.
Highlights
Aptamers are nucleic acid ligands that bind to target molecules [1,2]
To investigate whether the G4-forming DNAs bind to their protein products in vitro, we focused on the VEGFA, platelet-derived growth factor A (PDGFA), retinoblastoma 1 (RB1), and c-KIT genes
DNA aptamers with Kd values of 1.761027 M, 6.361029 M, and 4.461027 M, respectively; we did not obtain a c-KIT DNA aptamer. These results demonstrate that G4 promoter-derived aptamer selection (G4PAS) cannot be applicable to all proteins, DNA aptamers can be from the G4-forming promoter regions of the target proteins without Systematic Evolution of Ligands by EXponential enrichment (SELEX)
Summary
Aptamers are nucleic acid ligands that bind to target molecules [1,2]. Aptamers are selected from a random nucleic acid library in vitro, designated as Systematic Evolution of Ligands by EXponential enrichment (SELEX). In SELEX, oligonucleotides bound to the target are eluted and polymerase chain reaction (PCR)-amplified to prepare the round library. After several rounds of selection, the enriched library is sequenced and aptamers are identified. SELEX is an efficient method for screening aptamers, it sometimes fails to obtain aptamers because of PCR bias [6,7] and the limited diversity of the library used in experimental manipulation [8]. The development of a method for aptamer selection without SELEX is required
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