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.
Hygrophila difformis, a heterophyllous amphibious plant, develops serrated or dissected leaves when grown in terrestrial or submerged conditions, respectively. In this study, we tested whether submerged leaves and ethylene-induced leaves of the heterophyllous, amphibious plant H. difformis have improved photosynthetic ability under submerged conditions. Also, we investigated how this amphibious plant photosynthesizes underwater and whether a HCO3− transport system is present. We have analysed leaf morphology, measured underwater photosynthetic rates and HCO3− affinity in H. difformis to determine if there are differences in acclimation ability dependent on growth conditions: terrestrial, submerged, terrestrial treated with ethylene and submerged treated with an ethylene inhibitor. Moreover, we measured time courses for changes in leaf anatomical characteristics and underwater photosynthesis in terrestrial leaves after submersion. Compared with the leaves of terrestrially grown plants, leaf thickness of submerged plants was significantly thinner. The stomatal density on the abaxial surface of submerged leaves was also reduced, and submerged plants had a significantly higher O2 evolution rate. When the leaves of terrestrially grown plants were treated with ethylene, their leaf morphology and underwater photosynthesis increased to levels comparable to those of submerged leaves. Underwater photosynthesis of terrestrial leaves was significantly higher by 5 days after submersion. In contrast, leaf morphology did not change after submergence. Submerged leaves and submerged terrestrial leaves were able to use bicarbonate but submerged terrestrial leaves had an intermediate ability to use HCO3− that was between terrestrial leaves and submerged leaves. Ethoxyzolamide, an inhibitor of intracellular carbonic anhydrase, significantly inhibited underwater photosynthesis in submerged leaves. This amphibious plant acclimates to the submerged condition by changing leaf morphology and inducing a HCO3− utilizing system, two processes that are regulated by ethylene.
G-quadruplexes (G4s) are noncanonical DNA/RNA structures formed by guanine-rich sequences. Recently, G4s have been found not only in aptamers but also in the genomic DNA and transcribed RNA. In this study, we identified new RNA oligonucleotides working as aptamers by focusing on G4-forming RNAs located within the pre-mRNA. We showed that the G4 in the 5 1 UTR and first intron of VEGFA bound to the protein encoded in VEGFA gene, VEGF165, with high affinity. Moreover, G4-forming RNAs located within the PDGFA and the PDGFB introns bound to PDGF-AA and PDGF-BB, respectively, indicating that G4 in the pre-mRNA could be an aptamer. It had been reported that the putative G4-forming RNA sequences are located in some parts of most genes, thus our strategy for aptamer identification could be applicable to other proteins. It has been reported that some G4-forming RNAs in 5 1 UTRs are involved in translation control; however, G4-forming excised intronic RNA function has not been revealed previously. Therefore, these findings could not only contribute to the identification of RNA aptamers but also provide new insights into the biological functioning of G4-forming RNAs located within intronic RNA sequences.
We describe the selection of aptamers based on bioinformatics-based approaches without Systematic Evolution of Ligands by EXponential enrichment (SELEX). SELEX is a potent method; however, it is time intensive and the PCR-amplification step, which is essential step for SELEX, leads to the loss of good aptamers. We have developed an aptamer-screening method, G4 promoter-derived aptamer selection (G4PAS), and an aptamer-improving method, in silico maturation (ISM). They are based on in silico sequence selection and computer assisted directed evolution, respectively. In this study, we succeeded in identifying new aptamers against hepatocyte growth factor (HGF) by G4PAS as well as improving the specificity of the HGF aptamers by ISM. Using ISM improved the specificity of the aptamer for HGF by up to 45-fold in comparison with the original aptamer. These methods enable easy and efficient identification of good aptamers, and the combination of G4PAS with ISM can thus serve as a potent approach for aptamer identification. Biotechnol. Bioeng. 2017;114: 2196-2203. © 2017 Wiley Periodicals, Inc.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.