Kaori Tsukakoshi

Selection of DNA Aptamers That Recognize α-Synuclein Oligomers Using a Competitive Screening Method

α-Synuclein (α-syn) oligomers are considered major molecules responsible for the onset of Parkinsons disease and dementia with Lewy bodies. α-Syn oligomers thus serve as an important target for the development of drugs and diagnostic tests for neurodegenerative diseases. In this paper we report on the identification of DNA aptamers that bind to soluble α-syn oligomers. A competitive screening method based on aptamer blotting was used for the selection of α-syn oligomer-specific aptamers. This approach resulted in the identification of eight aptamers that specifically bind to α-syn oligomers among α-syn monomers, oligomers, and fibrils. Interestingly, the aptamers also bound to amyloid β oligomers, which are strongly associated with the development of Alzheimers disease. The results of this study support the hypothesis that amyloid oligomers share a common structure. Oligomer-binding aptamers may serve as powerful analytical tools for the design and development of drugs and diagnostic tests for neurodegenerative diseases.

Screening of DNA aptamer which binds to α-synuclein

Abstractα-Synuclein is a native, unfolded protein that causes several neurodegenerative diseases such as dementia with Lewy bodies and Parkinson’s disease. We have now identified the first DNA aptamers against α-synuclein using native PAGE applied to the SELEX method. We call this aptamer “M5-15”; it is the α-synuclein-bound aptamer and was isolated after four cycles of screening. M5-15 is composed of three stem-loop structures that may play an important role in the binding to α-synuclein. Moreover, M5-15 specifically binds to the α-synuclein monomer and oligomer. We expect that this aptamer will become a useful tool in α-synuclein analysis and diagnosis.

In silico maturation of binding‐specificity of DNA aptamers against Proteus mirabilis

Proteus mirabilis is a prominent cause of catheter‐associated urinary tract infections (CAUTIs) among patients undergoing long‐term bladder catheterization. There are currently no effective means of preventing P. mirabilis infections, and strategies for prophylaxis and rapid early diagnosis are urgently required. Aptamers offer significant potential for development of countermeasures against P. mirabilis CAUTI and are an ideal class of molecules for the development of diagnostics and therapeutics. Here we demonstrate the application of Cell‐SELEX to identify DNA aptamers that show high affinity for P. mirabilis. While the aptamers identified displayed high affinity for P. mirabilis cells in dot blotting assays, they also bound to other uropathogenic bacteria. To improve aptamer specificity for P. mirabilis, an in silico maturation (ISM) approach was employed. Two cycles of ISM allowed the identification of an aptamer showing 36% higher specificity, evaluated as a ratio of binding signal for P. mirabilis to that for Escherichia coli (also a cause of CAUTI and the most common urinary tract pathogen). Aptamers that specifically recognize P. mirabilis would have diagnostic and therapeutic values and constitute useful tools for studying membrane‐associated proteins in this organism. Biotechnol. Bioeng. 2013;110: 2573–2580.

Simultaneous improvement of specificity and affinity of aptamers against Streptococcus mutans by in silico maturation for biosensor development

In silico evolution with an in vitro system can facilitate the development of functional aptamers with high specificity and affinity. Although a general technique known as systematic evolution of ligand by exponential enrichment (SELEX) is an efficient method for aptamer selection, it sometimes fails to identify aptamers with sufficient binding properties. We have previously developed in silico maturation (ISM) to improve functions of aptamers based on genetic algorithms. ISM represents an intelligent exploitation of a random search within a defined sequence space to optimize aptamer sequences and improve their function of interest. Here we demonstrated a successful application of ISM of aptamers to simultaneously improve specificity and affinity for Streptococcus mutans with discovery of a core sequence, which was required to form a polymerized guanine quadruplex structure for target binding. We applied ISM to aptamers selected by whole‐cell SELEX and identified an aptamer with up to 16‐fold improvement in affinity compared to its parent aptamers, and specificity was increased to show 12‐fold more binding to S. mutans than to Lactobacillus acidophilus. Furthermore, we demonstrated a specific flow‐through detection of S. mutans at a concentration range of 1 × 105–108 CFU/mL using the evolved aptamer immobilized on gold colloids. Biotechnol. Bioeng. 2014;111: 454–461.

Improvement of the VEGF binding ability of DNA aptamers through in silico maturation and multimerization strategy

Aptamers are mainly selected by in vitro selection using random nucleic acid libraries. These aptamers have often shown insufficient affinity for biomedical applications. We improved DNA aptamer binding affinity for vascular endothelial growth factor (VEGF) through in silico maturation (ISM) and aptamer multimerization. ISM is one of a number of evolutionary approaches and aptamer multimerization is one of several semi-rational strategies to improve function. We first reselected VEGF-binding aptamers using a partially randomized DNA library and identified two aptamers with higher binding ability than that of a known aptamer. We conducted ISM using the re-selected aptamers to optimize the key loop sequences created by a three-way junction structure. After five ISM rounds, we identified aptamer 2G19 [dissociation constant (Kd), 52 nM] as a local optimum of the defined search space. We characterized the aptamer and found that a specific stem-loop structure was involved in aptamer VEGF recognition. To further improve its affinity for VEGF, we multimerized 2G19 or its stem-loop structure. The designed SL5-trivalent aptamer (Kd, 0.37 nM) with three binding motifs significantly increased binding affinity, representing a 500-fold improvement from systematic evolution of ligands by exponential enrichment-selected aptamers.

DNA aptamers against FokI nuclease domain for genome editing applications

Genome editing with site-specific nucleases (SSNs) can modify only the target gene and may be effective for gene therapy. The main limitation of genome editing for clinical use is off-target effects; excess SSNs in the cells and their longevity can contribute to off-target effects. Therefore, a controlled delivery system for SSNs is necessary. FokI nuclease domain (FokI) is a common DNA cleavage domain in zinc finger nuclease (ZFN) and transcription activator-like effector nuclease. Previously, we reported a zinc finger protein delivery system that combined aptamer-fused, double-strand oligonucleotides and nanoneedles. Here, we report the development of DNA aptamers that bind to the target molecules, with high affinity and specificity to the FokI. DNA aptamers were selected in six rounds of systematic evolution of ligands by exponential enrichment. Aptamers F6#8 and #71, which showed high binding affinity to FokI (Kd=82nM, 74nM each), showed resistance to nuclease activity itself and did not inhibit nuclease activity. We immobilized the ZFN-fused GFP to nanoneedles through these aptamers and inserted the nanoneedles into HEK293 cells. We observed the release of ZFN-fused GFP from the nanoneedles in the presence of cells. Therefore, these aptamers are useful for genome editing applications such as controlled delivery of SSNs.

Applying a riboregulator as a new chromosomal gene regulation tool for higher glycogen production in Synechocystis sp. PCC 6803

Cyanobacteria are one of the most attractive hosts for biofuel production; however, genetic approaches to regulate specific chromosomal genes in cyanobacteria remain limited. With the aim of developing a novel method to regulate chromosomal gene expression in cyanobacteria, we focused on riboregulatory technology. Riboregulators are composed of two RNA fragments whose interaction leads to target gene regulation with high specificity. In this study, we inserted a riboregulator sequence upstream of the chromosomal gene encoding AbrB-like transcriptional regulator, cyAbrB2, to investigate the utility of this tool. The inserted riboregulator was able to regulate cyabrB2 gene expression, with a high ON-OFF ratio up to approximately 50-fold. The transcription levels of several genes for which cyAbrB2 acts as a transcriptional upregulator were also decreased. Further, the cyAbrB2 expression-repressed mutant showed high glycogen accumulation, equivalent to that in the cyabrB2 deletion mutant (ΔcyabrB2). Phenotypic similarities between the cyabrB2 expression-repressed mutant and the ΔcyabrB2 mutant suggest that the riboregulator can potentially be used as a new chromosomal gene regulation tool in cyanobacteria.

Structural regulation by a G-quadruplex ligand increases binding abilities of G-quadruplex-forming aptamers

We report that a G-quadruplex-specific compound induced structural changes in G-quadruplex-forming DNA aptamers and stabilized the folded structures, making them suitable for targeted proteins and enhancing their binding properties.

Improving the induction fold of riboregulators for cyanobacteria

ABSTRACT Cyanobacteria are ideal cellular factories for biochemical production because of their ability to fix CO2 by photosynthesis and convert this molecule into biochemicals. Previously, we engineered a riboregulator that enables post-transcriptional gene regulation in the cyanobacterium Synechocystis sp. PCC 6803. Here, we improved the riboregulator by designing two RNA species, taRNA and crRNA, to enhance its induction fold. We inserted nucleotides into the crRNA loop to enhance intermolecular hybridization and successfully improved its induction fold. The engineered riboregulator exhibited a higher induction fold than the previously engineered riboregulator in both Escherichia coli and Synechocystis sp. PCC 6803. This improved riboregulator can be used to control gene expression over a wide dynamic range in cyanobacteria.

Non-label homogeneous protein detection based on laser interferometric photo-thermal displacement measurement using aptamers.

Photo-thermal displacement measurement by laser interferometry involves the measurement of temperature change caused by illumination of the sample. To develop a system of detecting unlabeled homogeneous proteins based on laser interferometric measurement of photo-thermal displacement, we studied the interaction between aptamers and their target molecules by using thrombin and the thrombin aptamer as a model target and ligand, respectively. Because of the energy consumed by aptamer-thrombin interactions, the signals obtained from solutions containing aptamer-thrombin mixtures varied depending on the thrombin concentration. We propose that this method involving the use of aptamers and photo-thermal displacement measurement will provide a biomolecular detection system for rapid diagnosis.