Analysis of the evolution of the thrombin-inhibiting DNA aptamers using a genetic algorithm

We previously identified a thrombin-inhibiting DNA aptamer that was presumed to form a G-quartet structure with a duplex. To investigate the importance of the sequences in the duplex region and to obtain aptamers with higher inhibitory activities, we randomized the sequences of the duplex region of this aptamer and carried out selection based on inhibitory activity using a genetic algorithm. This method consisted of selection via an inhibition assay, crossover, and mutation in silico. After two cycles, we obtained ligands with greater inhibitory activities than that of the original aptamer. In addition, the duplex sequences were found to contribute to the inhibitory activities of aptamers.     

Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin

Background

Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.

Principal Findings

Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.

Conclusion

We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.     

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.     

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.     

Detection system based on the conformational change in an aptamer and its application to simple bound/free separation

Aptamers are good molecular recognition elements for biosensors. Especially, their conformational change, which is induced by the binding to the target molecule, enables the development of several types of useful detection systems. We applied this property to bound/free separation, which is a crucial process for highly sensitive detection. We designed aptamers which change their conformation upon binding to the target molecule and thereby expose a single-strand bearing the complementary sequence to the capture probe immobilized onto the support. We named the designed aptamers "capturable aptamers" and the capture probe "capture DNA". Three capturable aptamers were designed based on the PrP aptamer, which binds to prion protein. One of these capturable aptamers was demonstrated to recognize prion protein and change its conformation upon binding to it. A detection system using this designed capturable aptamer for prion protein was developed. Capturable aptamers and capture DNA allow us to perform simple bound/free separation with only one target ligand.     

鳗弧菌(Vibrio anguillarum)核酸适配体的筛选及其结合蛋白的分离鉴定

目的 鳗弧菌(Vibrio anguillarum)是水产养殖中的重要条件致病菌,每年给水产养殖业造成巨大的经济损失,研究其致病机制、对其进行快速的检测鉴定是其病害防治的前提和基础.核酸适配体因其高亲和力、高特异性等多种优点,在微生物的靶标分析、检测鉴定以及致病机制等多个领域都呈现出较好的应用潜力.因此,筛选鳗弧菌的核...     

Aptameric enzyme subunit for homogeneous DNA sensing

We have developed an aptameric enzyme subunit (AES) which can detect the DNA in a homogeneous solution. The AES is an artificial enzyme subunit composed of an enzyme-inhibiting aptamer bearing a target-molecule binding site. We connected a probe DNA to a thrombin-inhibiting aptamer at its 5′ or 3′ end. The inhibitory activity of the thrombin-inhibiting aptamer bearing the probe DNA decreased compared to that of the original aptamer; however, it recovered upon hybridization with the target DNA. Using this AES, we were able to detect target DNAs by measuring the thrombin activity in a homogeneous solution. K. Ikebukuro and W. Yoshida have contributed equally to this work.     

Label-free homogeneous detection of immunoglobulin E by an aptameric enzyme subunit

We have developed an aptameric enzyme subunit (AES) for immunoglobulin E (IgE) sensing. AES is an artificial enzyme subunit constructed from two different aptamers and does not require any modification. Using the AES, the target molecule can be detected by measuring enzymatic activity in homogeneous solution. We connected IgE-binding aptamer and its complementary strand to split thrombin-inhibiting aptamer. The hybrid of these two oligonucleotides inhibited thrombin activity and it decreased in the presence of IgE. We were able to detect IgE by using this AES in homogeneous solution with a detection limit of 50 pmol.     

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.     

Developing aptamer probes for acute myelogenous leukemia detection and surface protein biomarker discovery

Background

The majority of patients with acute myelogenous leukemia (AML) still die of their disease. In order to improve survival rates in AML patients, new strategies are necessary to discover biomarkers for the detection and targeted therapy of AML. One of the advantages of the aptamer-based technology is the unique cell-based selection process, which allows us to efficiently select for cell-specific aptamers without knowing which target molecules are present on the cell surface.

Methods

The NB4 AML cell line was used as the target cell population for selecting single stranded DNA aptamers. After determining the affinity of selected aptamers to leukocytes, the aptamers were used to phenotype human bone marrow leukocytes and AML cells in clinical specimens. Then a biotin-labelled aptamer was used to enrich and identify its target surface protein.

Results

Three new aptamers were characterized from the selected aptamer pools (JH6, JH19, and K19). All of them can selectively recognize myeloid cells with Kd in the low nanomole range (2.77 to 12.37 nM). The target of the biotin-labelled K19 aptamer probe was identified as Siglec-5, a surface membrane protein in low abundance whose expression can serve as a biomarker of granulocytic maturation and be used to phenotype AML. More importantly, Siglec-5 expression can be used to detect low concentrations of AML cells in human bone marrow specimens, and functions as a potential target for leukemic therapy.

Conclusions

We have demonstrated a pipeline approach for developing single stranded DNA aptamer probes, phenotyping AML cells in clinical specimens, and then identifying the aptamer-recognized target protein. The developed aptamer probes and identified Siglec-5 protein may potentially be used for leukemic cell detection and therapy in our future clinical practice.     

Screening of DNA Aptamer Against Mouse Prion Protein by Competitive Selection

Prion disease is a neurodegenerative disorder, in which the normal prion protein (PrP) changes structurally into an abnormal form and accumulates in the brain. There is a great demand for the development of a viable approach to diagnosis and therapy. Not only has the ligand against PrP been used for diagnosis, but it has also become a promising tool for therapy, as an antibody. Aptamers are a novel type of ligand composed of nucleic acids. DNA aptamers in particular have many advantages over antibodies. Therefore, we tried to isolate the DNA aptamer for mouse PrP. We developed a competitive selection method and tried to screen the DNA aptamer with it. In the fourth round of selection, several clones of the aptamer with an affinity to PrP were enriched, and clone 4–9 showed the highest affinity of all. The investigation by aptamer blotting and Western blotting showed that clone 4–9 was specifically able to recognize both α-PrP and β-PrP. Moreover, it was indicated that clone 4–9 could recognize the flexible region of the N-terminal domain of PrP. These characteristics suggest that clone 4–9 might be a useful tool in prion-disease diagnosis and research.Key Words: aptamer, DNA, SELEX, competitive selection, prion protein, β-PrP, detection     

Sialyllactose-binding modified DNA aptamer bearing additional functionality by SELEX

We produced a novel cationic-charged modified DNA aptamer for sialyllactose that is a ubiquitous component of the cell surface responsible for the infection of several viruses by using the magnetic-particle-based SELEX method. After 13 rounds of selection we selected 22 clones as sialyllactose-binding DNA aptamers composed of several modified thymidines. The DNA aptamers could form a three-way junction structure that likely forms a binding site for siallyllactose. The three-way junction structure contains several modified thymidines bearing a positively-charged amino group at the C5 position, which could enhance the binding ability for silalyllactose which has a negatively-charged carboxyl group. The dissociation constant of the aptamer that showed the strongest sialyllactose-binding ability among the clones of the aptamers was 4.9 microM.     

Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to develop DNA ligands (aptamers) to cholera whole toxin and staphylococcal enterotoxin B (SEB). Affinity selection of aptamers was accomplished by conjugating the biotoxins to tosyl-activated magnetic beads. The use of magnetic beads reduces the volumes needed to perform aptamer selection, thus obviating alcohol precipitation and allowing direct PCR amplification from the bead surface. Following five rounds of SELEX, 5'-biotinylated aptamers were bound to streptavidin-coated magnetic beads and used for the detection of ruthenium trisbypyridine [Ru(bpy)3(2+)]-labeled cholera toxin and SEB by an electrochemiluminescence methodology. A comparison of control (double-stranded) aptamer binding was made with aptamers that were heat denatured at 96 degrees C (single-stranded) and allowed to cool (conform) in the presence of biotoxin-conjugated magnetic beads. Results suggest that control aptamers performed equally well when compared to heat-denatured DNA aptamers in the cholera toxin electrochemiluminescence assay and a colorimetric microplate assay employing peroxidase-labeled cholera toxin and 5'-amino terminated aptamers conjugated to N-oxysuccinimide-activated microtiter wells. Interestingly, however, in the SEB electrochemiluminescence assay, double-stranded aptamers exceeded the performance of single-stranded aptamers. The detection limits of all aptamer assays were in the low nanogram to low picogram ranges.     

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications

A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G–C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF₁₆₅ unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF₁₆₅ RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF₁₆₅. Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF₁₆₅ and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF₁₆₅ DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF₁₆₅.     

A hotspot for enhancing insulin receptor activation revealed by a conformation-specific allosteric aptamer

Aptamers are single-stranded oligonucleotides that bind to a specific target with high affinity, and are widely applied in biomedical diagnostics and drug development. However, the use of aptamers has largely been limited to simple binders or inhibitors that interfere with the function of a target protein. Here, we show that an aptamer can also act as a positive allosteric modulator that enhances the activation of a receptor by stabilizing the binding of a ligand to that receptor. We developed an aptamer, named IR-A43, which binds to the insulin receptor, and confirmed that IR-A43 and insulin bind to the insulin receptor with mutual positive cooperativity. IR-A43 alone is inactive, but, in the presence of insulin, it potentiates autophosphorylation and downstream signaling of the insulin receptor. By using the species-specific activity of IR-A43 at the human insulin receptor, we demonstrate that residue Q272 in the cysteine-rich domain is directly involved in the insulin-enhancing activity of IR-A43. Therefore, we propose that the region containing residue Q272 is a hotspot that can be used to enhance insulin receptor activation. Moreover, our study implies that aptamers are promising reagents for the development of allosteric modulators that discriminate a specific conformation of a target receptor.     

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.     

Screening of DNA Aptamer Against Mouse Prion Protein by Competitive Selection

Prion disease is a neurodegenerative disorder, in which the normal prion protein (PrP) changes structurally into an abnormal form and accumulates in the brain. There is a great demand for the development of a viable approach to diagnosis and therapy. Not only has the ligand against PrP been used for diagnosis, but it has also become a promising tool for therapy, as an antibody. Aptamers are a novel type of ligand composed of nucleic acids. DNA aptamers in particular have many advantages over antibodies. Therefore, we tried to isolate the DNA aptamer for mouse PrP. We developed a competitive selection method and tried to screen the DNA aptamer with it. In the fourth round of selection, several clones of the aptamer with an affinity to PrP were enriched, and clone 4-9 showed the highest affinity of all. The investigation by aptamer blotting and Western blotting showed that clone 4-9 was specifically able to recognize both α-PrP and β-PrP. Moreover, it was indicated that clone 4-9 could recognize the flexible region of the N-terminal domain of PrP. These characteristics suggest that clone 4-9 might be a useful tool in prion-disease diagnosis and research.     

Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer

A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15–0.50 kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by T_m versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation.     

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets (“apatopes”) with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer–aptamer, aptamer–nonaptamer biomacromolecules (siRNAs, proteins) and aptamer–nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.     

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.