Aptamer Selection Based on G4-Forming Promoter Region

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.     

A colorimetric detection method of pesticide acetamiprid by fine-tuning aptamer length

This work investigates the effect of shortening aptamer sequences on the colorimetric detection of acetamiprid using aptamer-wrapped gold nanoparticles (AuNPs). Truncated 37-mer and 25-mer aptamers were generated by deleting excess flanking nucleotides from parental 49-mer acetamiprid-target aptamer. In comparing the responses of the three sequences, truncated aptamers did not improve the ability to discriminate against other tested pesticides. However, comparison between 49-mer and other shorter aptamers showed that shortening aptamer sequences through removing excess flanking nucleotides outsides of binding region improved colorimetric sensitivity for acetamiprid by 3.3 fold. Due to excess bases, the target-bound aptamer might still adhere to AuNPs, resulting in incomplete dissociation of aptamer from AuNPs and therefore the suppression of aggregation responses. This work provides further insight to the effects of aptamer structure on detection of the target, as well as a method by fine-tuning aptamer length for rapid detection of pesticide residues in environments or food.     

G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin''s peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.     

Designing Anti-Influenza Aptamers: Novel Quantitative Structure Activity Relationship Approach Gives Insights into Aptamer – Virus Interaction

This study describes the development of aptamers as a therapy against influenza virus infection. Aptamers are oligonucleotides (like ssDNA or RNA) that are capable of binding to a variety of molecular targets with high affinity and specificity. We have studied the ssDNA aptamer BV02, which was designed to inhibit influenza infection by targeting the hemagglutinin viral protein, a protein that facilitates the first stage of the virus’ infection. While testing other aptamers and during lead optimization, we realized that the dominant characteristics that determine the aptamer’s binding to the influenza virus may not necessarily be sequence-specific, as with other known aptamers, but rather depend on general 2D structural motifs. We adopted QSAR (quantitative structure activity relationship) tool and developed computational algorithm that correlate six calculated structural and physicochemical properties to the aptamers’ binding affinity to the virus. The QSAR study provided us with a predictive tool of the binding potential of an aptamer to the influenza virus. The correlation between the calculated and actual binding was R² = 0.702 for the training set, and R² = 0.66 for the independent test set. Moreover, in the test set the model’s sensitivity was 89%, and the specificity was 87%, in selecting aptamers with enhanced viral binding. The most important properties that positively correlated with the aptamer’s binding were the aptamer length, 2D-loops and repeating sequences of C nucleotides. Based on the structure-activity study, we have managed to produce aptamers having viral affinity that was more than 20 times higher than that of the original BV02 aptamer. Further testing of influenza infection in cell culture and animal models yielded aptamers with 10 to 15 times greater anti-viral activity than the BV02 aptamer. Our insights concerning the mechanism of action and the structural and physicochemical properties that govern the interaction with the influenza virus are discussed.     

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.     

Screening and Characterization of a Novel RNA Aptamer That Specifically Binds to Human Prostatic Acid Phosphatase and Human Prostate Cancer Cells

Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2′-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2′-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer.     

Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 10¹⁶ different RNA or DNA sequences by 5–10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2′-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers.     

Development of DNA Aptamers to a Foot-and-Mouth Disease Peptide for Competitive FRET-Based Detection

We sought to develop a novel competitive fluorescence resonance energy transfer (FRET)-aptamer-based strategy for detection of foot-and-mouth (FMD) disease within minutes. A 14-amino-acid peptide from the VP1 structural protein, which is conserved among 16 strains of O-serotype FMD virus, was synthesized and labeled with Black Hole Quencher-2 (BHQ-2) dye. Polyclonal FMD DNA aptamers were labeled with Alexa Fluor 546-14-dUTP by polymerase chain reaction and allowed to bind the BHQ-2-peptide conjugate. Following purification of the FRET–aptamer–peptide complex, a “lights off” response was observed within 10 minutes and was sensitive to a level of 25–250 ng/mL of FMD peptide. Ten candidate aptamers were sequenced from the polyclonal family. The aptamer candidates were screened in an enzyme-based plate assay. A high- and low-affinity aptamer candidate were each labeled with Alexa Fluor 546-14-dUTP by asymmetric polymerase chain reaction and used in the competitive FRET assay, but neither matched the sensitivity of the polyclonal FRET response, indicating the need for further screening of the aptamer library.     

Selection of aptamers against inactive Vibrio alginolyticus and application in a qualitative detection assay

Aptamers against inactive Vibrio alginolyticus were selected from an 82-nt ssDNA random library by systematic evolution of ligands by exponential enrichment. After 15 rounds of selection, the final pool of aptamers was highly specific for inactivated V. alginolyticus and had a dissociation constant of 27.5 ± 9.2 nM. Using these aptamers and PCR, V. alginolyticus could be detected at 100 cells/ml. Sequencing of the final pool of aptamers revealed that some sequences, termed high-frequency aptamers, appeared more than once; these may be of practical application. All sequences obtained were divided into nine families according to their homology tree, some conserved sequences were also found in each of the six families. One sequence was found in significant proportions of the aptamers, suggesting that this conserved sequence might be important for forming the three-dimensional aptamer structure.     

Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection

BackgroundThe broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers.

Methodology/Principal Findings

We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs.

Conclusions and Significance

We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.     

Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris

Mature human growth hormone (hGH) cDNA was cloned by homologous recombination into the yeast Pichia pastoris genome. The hGH gene expression was placed under the control of the methanol-inducible alcohol oxidase 1 (AOX1) gene promoter and the Saccharomyces cerevisiae -factor signal sequence to direct the secretion of recombinant human growth hormone (rhGH) into the growth medium. O₂-limited induction of recombinant yeast strains in shake tubes with 3 ml of culture medium produced up to 11 mg rhGH l^–1, while high cell density cultures using a 2-l bioreactor produced about 49 mg rhGH l^–1 achieving 40% of total protein of the culture medium supernatant.     

In vitro selection and characterization of cellulose-binding DNA aptamers

Many nucleic acid enzymes and aptamers have modular architectures that allow them to retain their functions when combined with other nucleotide sequences. This modular function facilitates the engineering of RNAs and DNAs that have more complex functions. We sought to create new DNA aptamers that bind cellulose to provide a module for immobilizing DNAs. Cellulose has been used in a variety of applications ranging from coatings and films to pharmaceutical preparations, and therefore DNA aptamers that bind cellulose might enable new applications. We used in vitro selection to isolate aptamers from a pool of random-sequence DNAs and subjected two distinct clones to additional rounds of mutagenesis and selection. One aptamer (CELAPT 14) was chosen for sequence minimization and more detailed biochemical analysis. CELAPT 14 aptamer variants exhibit robust binding both to cellulose powder and paper. Also, an allosteric aptamer construct was engineered that exhibits ATP-mediated cellulose binding during paper chromatography.     

Designing a new dimerized anti human TNF-α aptamer with blocking activity

The human tumor necrosis factor α (hTNF-α) is an important pro-inflammatory cytokine which plays critical roles in inflammatory diseases such as rheumatoid arthritis (RA). The anti-TNF-α proteins can reduce symptoms of RA. Due to limitations of protein-based therapies, it is necessary to find new anti-TNF-α agents instead of common anti-TNF-α proteins. Therefore, the aim of the current study was to identify a new DNA aptamer with anti-hTNF-α activity. The protein systematic evolution of ligands by exponential enrichment (SELEX) process was used for identifying DNA aptamers. Anti-hTNF-α aptamers were selected using dot blot, real-time PCR, and in vitro inhibitory assay. The selected aptamers were truncated in two steps, and finally, a dimer aptamer was constructed from different selected truncates to improve their inhibitory effect. Also, Etanercept was used as a positive control to inhibit TNF-α, in comparison to the designed aptamers. After 11 rounds, four aptamers with anti-hTNF-α inhibitory effect were identified. The truncation and dimerization strategy revealed a new dimer aptamer with 67 nM K_d, which has 40% inhibitory effect compared with Etanercept (60%). Overall, the dimerization and truncation aptamers could improve its activity. With regard to the several limitations of anti-TNF-α proteins therapies including immunogenicity, side effects, and cost-intensive, a new designed anti-hTNF-α dimer aptamer could be considered as a potential therapeutic and/or diagnostic agent for hTNF-α-related disorders.     

Predicting candidate genomic sequences that correspond to synthetic functional RNA motifs

Riboswitches and RNA interference are important emerging mechanisms found in many organisms to control gene expression. To enhance our understanding of such RNA roles, finding small regulatory motifs in genomes presents a challenge on a wide scale. Many simple functional RNA motifs have been found by in vitro selection experiments, which produce synthetic target-binding aptamers as well as catalytic RNAs, including the hammerhead ribozyme. Motivated by the prediction of Piganeau and Schroeder [(2003) Chem. Biol., 10, 103–104] that synthetic RNAs may have natural counterparts, we develop and apply an efficient computational protocol for identifying aptamer-like motifs in genomes. We define motifs from the sequence and structural information of synthetic aptamers, search for sequences in genomes that will produce motif matches, and then evaluate the structural stability and statistical significance of the potential hits. Our application to aptamers for streptomycin, chloramphenicol, neomycin B and ATP identifies 37 candidate sequences (in coding and non-coding regions) that fold to the target aptamer structures in bacterial and archaeal genomes. Further energetic screening reveals that several candidates exhibit energetic properties and sequence conservation patterns that are characteristic of functional motifs. Besides providing candidates for experimental testing, our computational protocol offers an avenue for expanding natural RNA's functional repertoire.     

Probing the limits of aptamer affinity with a microfluidic SELEX platform

Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K_d) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K_d of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K_d.     

Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing

We selected DNA aptamers against insulin and developed an aptameric enzyme subunit (AES) for insulin sensing. The insulin-binding aptamers were identified from a single-strand DNA library which was expected to form various kinds of G-quartet structures. In vitro selection was carried out by means of aptamer blotting, which visualizes the oligonucleotides binding to the target protein at each round. After the 6th round of selection, insulin-binding aptamers were identified. These identified insulin-binding aptamers had a higher binding ability than the insulin-linked polymorphic region (ILPR) oligonucleotide, which can be called a "natural" insulin-binding DNA aptamer. The circular-dichroism (CD) spectrum measurement of the identified insulin-binding DNA aptamers indicated that the aptamers would fold into a G-quartet structure. We also developed an AES by connecting the best identified insulin-binding aptamer with the thrombin-inhibiting aptamer. Using this AES, we were able to detect insulin by measuring the thrombin enzymatic activity without bound/free separation.     

Selection of aptamers for aflatoxin M1 and their characterization

In the present work, aptamers against aflatoxin M1 and aflatoxin B1 were generated and tested for creating proof of principle of recognition of aflatoxin M1 by generated aptamers. The aptamers were selected through the process referred as systematic evolution of ligands by exponential enrichment. A total of 41 different aptamer (36 aptamers for aflatoxin M1 and 5 for aflatoxin B1) sequences were obtained. The determination of dissociation constant (K_d) values revealed that aptamers generated against aflatoxin M1 exhibited K_d values in the range of 35–1515 nM. Selected aptamers were grouped on the basis of the presence of common motifs or G‐quadruplex. We find it interesting that one aptamer with no conserved motif or G‐quadruplex had lowest K_d value (K_d = 35 nM). This structural motif is very distinct from motifs present in other aptamers. The K_d values of selected aptamers for aflatoxin B1 were in the range of 96–221 nM. One aptamer from each group was further tested for its ability to be used in aptasensor. The aptamer recognized aflatoxin M1 as indicated by color change (red to purple or blue) of aptamer‐coated gold nanoparticles in the presence of 250–500 nM aflatoxin M1. The aptamers can be used in developing methods for detection/estimation/separation of aflatoxin or antidote for aflatoxin toxicity. Copyright © 2014 John Wiley & Sons, Ltd.