RNAs fold into specific complex three-dimensional structures to provide catalytic and molecular recognition abilities. These functional RNAs have been identified in both natural and artificial RNAs [1
]. Artificial functional RNAs that possess molecular recognition ability are known as RNA aptamers, and such aptamers can be selected from random RNA libraries by systematic evolution of ligands by exponential enrichment (SELEX) [2
]. The chemically modified RNA aptamers are valuable as drug and molecular recognition elements in biosensors [4
]. Indeed, the chemically modified RNA aptamers against vascular endothelial growth factor 165 (VEGF165) have been approved for the treatment of neovascular age-related macular degeneration [5
]. Moreover, RNA aptamers have been utilized to develop ligand-inducible gene expression systems in synthetic biology [6
SELEX enables the identification of aptamers against small molecules, peptides, proteins, and whole cells, including non-immunogenic and toxic targets [9
]. Modified SELEX methods have been developed to efficiently select aptamers [10
]. However, SELEX is labor-intensive and generally requires several months to obtain aptamers. Non-SELEX aptamer identification methods such as the in silico
selection of aptamers have been reported [11
]. Although such virtual screening methods are potentially powerful, an improvement in the system is required to enable the selection of an appropriate aptamer without additional in vitro
selection. Aptamers can also be identified from genomic information without in vitro
selection. Riboswitches, which sense metabolites to control gene expression, translation, splicing, and RNA stability, are known to be natural aptamers [1
]. Riboswitch candidates have been identified by bioinformatic searches; however, these are limited to aptamers against small molecules [13
We have previously reported that G4-forming DNAs that are located within the gene promoter region of a target protein may act as DNA aptamers against the target proteins [14
]. The DNA aptamer identification method was designated as G4 promoter-derived aptamer selection (G4PAS). Using G4PAS, DNA aptamers against VEGF165, platelet-derived growth factor-AA (PDGF-AA), and retinoblastoma 1 (RB1) were identified. These aptamers bound to their respective target proteins with high affinity. In addition, the PDGF-AA and RB1 aptamers bound to VEGF165. However, the PDGF-AA and RB1 aptamers did not bind to VEGF121, which does not possess a heparin-binding domain, indicating that they bind to VEGF165 probably due to recognition of the heparin-binding domain.
We considered the G4 structure as a good scaffold for target protein recognition because G4 has twice the negative charge density of double helices [15
] to interact with the cationic domain of the protein. Moreover, we have suggested that G4 tends to preferentially bind to the β-structures of proteins [16
]. Therefore, we investigated whether G4-forming RNA aptamers could be obtained from the transcribed RNA of target proteins. It has been reported that potential G4-forming sequences are enriched at the transcription start site, the 5′ UTR, and in the first intron, which is on the non-template strand in the human genome [17
]. This suggests that G4-forming RNAs mainly exist within the 5′ UTR and the first intronic region of RNA. We chose to use VEGF165, PDGF-AA, and PDGF-BB as target proteins since each of them contains a heparin-binding domain, and G4-forming sequences are enriched in these genes. Analyses of the binding of target proteins to the synthetic G4-forming RNAs located within the 5′ UTR or the first intronic RNA region of these target genes were performed in vitro
3. Experimental Section
VEGF165, PDGF-AA, and PDGF-BB were purchased from R & D systems (Minneapolis, MN, USA). Human alpha-Thrombin was purchased from Haematologic Technologies Inc. (Essex, VT, USA). All RNAs were purchased from Greiner Japan (Tokyo, Japan) or Hokkaido System Science (Hokkaido, Japan).
3.2. Identification of Putative G4-Forming RNAs
Transcribed RNA sequences of VEGFA
, and PDGFB
were obtained from the UCSC genome browser [36
] and putative G4-forming RNAs located on the transcribed RNAs were identified by the QGRS Mapper [18
]. The synthesized RNAs were dissolved as 100 μM stock solutions in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
3.3. Electromobility Shift Assay (EMSA)
First, the chemically synthesized RNA oligonucleotides were labeled with fluorescein dye using the 5′ EndTag™ nucleic acid labeling system (Vector Laboratories, Burlingame, CA, USA). 0.8 nmol RNAs were used for fluorescein labeling and the labeling procedures were performed according to a prescribed protocol. After that, the ethanol precipitated RNAs were dissolved in 10 μL TE buffer.
Next EMSA was performed for evaluation of binding ability of RNAs. Fluorescein labeled RNAs were diluted into 2 μM in Tris-HCl potassium chloride buffer (50 mM Tris-HCl, with 100 mM KCl, pH 7.5) and folded at 65 °C for five min and then allowed to cool to room temperature for 30 min. Heat treated RNAs (f.c. 1 μM) and several concentrations of VEGF165 (f.c. 0.25, 0.5, 1, 2 μM) were mixed in Tris-HCl potassium chloride buffer. For inhibition of RNA degradation, 1 U of RNase OUT (Invitrogen, Carlsbad, CA, USA) was mixed into the samples then the mixtures were incubated at room temperature for 30 min. After incubation, ten microliters of the mixtures were electrophoresed on 15% polyacrylamide gel in TBE buffer, followed by fluorescein scanning the gel using Typhoon8600 (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
3.4. Surface Plasmon Resonance (SPR) Assay
The binding affinities of the G4-forming RNAs for VEGF165, PDGF-AA, and PDGF-BB were analyzed at 25 °C on a Biacore T200 instrument (GE Healthcare). VEGF165 (in 10 mM acetate buffer, pH 6.0), PDGF-AA (in 10 mM HEPES, pH 7.0), and PDGF-BB (in 10 mM HEPES, pH 7.0) were immobilized on a CM5 sensor chip (GE Healthcare) by the amine coupling procedure, and the RNAs were injected over the surface. Prior to use, all RNAs were denatured in PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) at 65 °C for five min, and then allowed to cool to room temperature for 30 min. PBS served as both the running and aptamer dilution buffers. SPR signals were used for the construction of the dissociation constant (Kd value), and Kd was estimated by curve fitting using BIAevaluation software (GE Healthcare).
3.5. Circular Dichroism (CD) Spectroscopy
Circular dichroism (CD) spectra were recorded on a Jasco-820 spectropolarimeter (JASCO; Tokyo, Japan) using a quartz cell of 10 mm optical path length (Agilent Technologies, Santa Clara, CA, USA) and an instrument scanning speed of 500 nm/min. All RNA samples were diluted to 2 μM in PBS buffer. These RNA samples were then folded by heat treatment as described above. The CD spectra are representations of 10 averaged scans taken at 25 °C.
In this study, we identified new RNA oligonucleotides that formed G4 located within the 5′ UTR and first intron of VEGFA bind to VEGF165 with high affinity. Moreover, G4-forming RNAs located within PDGFA and PDGFB introns bound to PDGF-AA and PDGF-BB, respectively. Mutation analysis of G4-forming RNAs-VEGF165 interaction indicated that the G4 structure was important for binding to VEGF165. These results indicated that G4s in the pre-mRNA would be aptamers against the target protein that is encoded in the pre-mRNA. The potential G4-forming RNA sequences are located in RNA regions of many of the kinds of genes and the physiological function of the G4 RNAs has not been revealed. Taken together, our present research provides not only the methodology of G4-forming RNA aptamer identification, but also new insights on the physiological function of the G4-forming RNAs.