In Situ Electrical Monitoring of Methylated DNA Based on Its Conformational Change to G-Quadruplex Using a Solution-Gated Field-Effect Transistor.

Methylated DNA is not only a diagnostic but also a prognostic biomarker for early-stage cancer. However, sodium bisulfite sequencing as a "gold standard" method for detection of methylation markers has some drawbacks such as its time-consuming and labor-intensive procedures. Therefore, simple and reliable methods are required to analyze DNA sequences with or without methylated residues. Herein, we propose a simple and direct method for detecting DNA methylation through its conformation transition to G-quadruplex using a solution-gated field-effect transistor (SG-FET) without using labeled materials. The BCL-2 gene, which is involved in the development of various human tumors, contains G-rich segments and undergoes a conformational change to G-quadruplex depending on the K+ concentration. Stacked G-quadruplex strands move close to the SG-FET sensor surface, resulting in large electrical signals based on intrinsic molecular charges. In addition, a dense hydrophilic polymer brush is grafted using surface-initiated atom transfer radical polymerization onto the SG-FET sensor surface to reduce electrical noise based on nonspecific adsorption of interfering species. In particular, control of the polymer brush thickness induces electrical signals based on DNA molecular charges in the diffusion layer, according to the Debye length limit. A platform based on the SG-FET sensor with a well-defined polymer brush is suitable for in situ monitoring of methylated DNA and realizes a point-of-care device with a high signal-to-noise ratio and without the requirement for additional processes such as bisulfite conversion and polymerase chain reaction.

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands.

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.

Methods for Improving Aptamer Binding Affinity.

Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers.

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 × 10(5) -10(8) CFU/mL using the evolved aptamer immobilized on gold colloids.

A novel electrochemical biosensing method with double-layered polymer brush modified electrode.

We developed a novel electrochemical biosensor electrode that has a potential to reduce background noise for which we constructed an original conductive substrate modified with a double-layered polymer brush structure that is water impermeable and can control biomolecules adsorption/desorption. In this study, a hydrophobic poly(tert-butyl methacrylate) brush layer was prepared on a gold electrode, and then, the tert-butyl group near the outermost surface was dissociated by the acid treatment to obtain a hydrophilic carboxy group, thereby fabricating a conductive substrate with the double-layered polymer brush structure. Formation of the double-layered polymer brush structure was indicated by surface wettability and optical analyses. The potential difference and hydrogen ion concentration, which is a typical parameter of the surrounding environment, were linearly correlated with the gold electrode having a double-layered polymer brush structure with carboxyl groups. However, there was no correlation on gold electrodes with self-assembled monolayers presenting carboxy groups. It is considered that the pH responsiveness of the carboxy groups on the outermost surface could be exhibited remarkably because the charge state in the vicinity of the surface became constant due to the hydrophobic polymer brush layer having a certain thickness. The target DNA could be captured more efficiently at the probe DNA-immobilized electrode with the double-layered polymer brush structure than when using COOH-SAM. This is the first report of the application of the double-layered polymer brush structure for the electrochemical biosensing, and it will be an excellent surface modification method to reduce background noise.

Selection of DNA aptamers against VEGF165 using a protein competitor and the aptamer blotting method.

Two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF(165)) were identified. In the screening process, another protein was used as the competitor to isolate those aptamers that have high specificity for the target. In addition, we evaluated the affinities of the enriched library by means of aptamer blotting. The isolated aptamers bound to VEGF(165) with a K(d) value in the range of a few hundred nanomoles, and did not bind to the competitor. This selection method enabled us to efficiently select the specific aptamers against the target protein. These specific aptamers would be useful sensor elements for cancer diagnosis.

Improvement of Aptamer Affinity by Dimerization.

To increase the affinities of aptamers for their targets, we designed an aptamerdimer for thrombin and VEGF. This design is based on the avidity of the antibody, whichenables the aptamer to connect easily since it is a single-strand nucleic acid. In this study,we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer.Each aptamer recognizes a different part of the thrombin molecule, and the aptamer dimerhas a Kd value which is 1/10 of that of the monomers from which it is composed. Also, thedesigned aptamer dimer has higher inhibitory activity than the reported (15-mer) thrombin-inhibiting aptamer. Additionally, we connected together two identical aptamers againstvascular endothelial growth factor (VEGF165), which is a homodimeric protein. As in thecase of the anti-thrombin aptamer, the dimeric anti-VEGF aptamer had a much lower Kd value than that of the monomer. This study demonstrated that the dimerization of aptamerseffectively improves the affinities of those aptamers for their targets.

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 and characterization of DNA aptamers against VEGF165 with aptamer blotting method and its application.

We have isolated two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF165). To select aptamers that have high specificity for VEGF165, we used another protein as a competitor in the SELEX process. In addition, we evaluated the screened library with aptamer blotting. Isolated aptamers bound to VEGF165 with Kd in a few hundred-nanomolar ranges, and did not bind to a competitor. This selection method allowed us to select the specific aptamers against a target protein efficiently. Furthermore, isolated aptamers did not bind to VEGF121. These specific DNA aptamers against VEGF165 would be a useful sensing element for cancer diagnosis.

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 alpha-PrP and beta-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.