Development of a Low-Molecular-Weight Aß42 Detection System Using a Enzyme-Linked Peptide Assay.

Alzheimer's disease (AD) is a degenerative brain disease that is the most common cause of dementia. The incidence of AD is rapidly rising because of the aging of the world population. Because AD is presently incurable, early diagnosis is very important. The disease is characterized by pathological changes such as deposition of senile plaques and decreased concentration of the amyloid-beta 42 (Aß42) peptide in the cerebrospinal fluid (CSF). The concentration of Aß42 in the CSF is a well-studied AD biomarker. The specific peptide probe was screened through four rounds of biopanning, which included the phage display process. The screened peptide showed strong binding affinity in the micromolar range, and the enzyme-linked peptide assay was optimized using the peptide we developed. This diagnostic method showed specificity toward Aß42 in the presence of other proteins. The peptide-binding site was also estimated using molecular docking analysis. Finally, the diagnostic method we developed could significantly distinguish patients who were classified based on amyloid PET images.

Development of a Novel ssDNA Sequence for a Glycated Human Serum Albumin and Construction of a Simple Aptasensor System Based on Reduced Graphene Oxide (rGO).

Diabetes is one of the top 10 global causes of death. About one in 11 global adults have diabetes. As the disease progresses, the mortality rate increases, and complications can develop. Thus, early detection and effective management of diabetes are especially important. Herein, we present a novel glycated human serum albumin (GHSA) aptamer, i.e., GABAS-01, which has high affinity and specificity. The aptamer was selected by reduced graphene oxide-based systematic evolution of ligands by exponential enrichement (rGO-based SELEX) against GHSA. After five rounds of selection through gradually harsher conditions, GABAS-01 with high affinity and specificity for the target was obtained. GABAS-01 was labeled by FAM at the 5'-end and characterized by measuring the recovery of a fluorescence signal that is the result of fluorescence quenching effect of rGO. As a result, GABAS-01 had low-nanomolar Kd values of 1.748 ± 0.227 nM and showed a low limit of detection of 16.40 µg/mL against GHSA. This result shows the potential application of GABAS-01 as an effective on-site detection probe of GHSA. In addition, these properties of GABAS-01 are expected to contribute to detection of GHSA in diagnostic fields.

Development of peptide aptamers as alternatives for antibody in the detection of amyloid-beta 42 aggregates.

Alzheimer's disease (AD) causes cognitive impairment and serious social isolation. However, there are no effective treatments and even no established confirmatory diagnostic tools for the disease. Amyloid beta (Aß) aggregation in the brain is the best-known pathognomonic mechanism of AD, so various methods for Aß detection have been developed for the diagnosis of this disease. We synthesized two novel, ultra-sensitive peptide probes specialized in detecting Aß aggregates, and examined their potential for future diagnostic application. The peptides are produced through phage high-throughput screening (HTS) and amplified through a serial process called biopanning, which is a repeating method of elution and amplification of probes. We picked phages specific for amyloid from two kinds of phage display. The synthesized peptides were confirmed to have excellent binding affinity to Aß aggregates, by immunohistochemical staining and western blotting using the brains of 3X transgenic (Tg) AD mice at different stages (5-7, 12-17 months old) of AD severity. In the present study, it was confirmed that newly developed amyloid-binding peptides could be used as novel probes for the detection of Aß aggregates, which can be used for clinical diagnosis of AD in the future.

Development of ssDNA Aptamers for Diagnosis and Inhibition of the Highly Pathogenic Avian Influenza Virus Subtype H5N1.

Avian influenza (AI) has severely affected the poultry industry worldwide and has caused the deaths of millions of birds. Highly pathogenic avian influenza virus is characterized by high mortality and the ability to transmit from birds to humans. Early diagnosis is difficult because of the variation in pathogenicity and the genetic diversity between virus subtypes. Therefore, development of a sensitive and accurate diagnostic system is an urgent priority. We developed ssDNA aptamer probes to detect AI viruses. Through seven rounds of SELEX to search for a probe specific to the highly pathogenic AI virus subtype H5N1, we identified 16 binding aptamers and selected two with the highest binding frequency. These two aptamers had strong binding affinities and low detection limits. We found that they could bind more specifically to H5N1, as compared to other subtypes. Furthermore, these aptamers inhibited hemagglutination, which is caused by the virus surface protein hemagglutinin. Our results indicate that our screened aptamers are effective molecular probes for diagnosing H5N1 and can be used as therapeutic agents to inhibit viral surface proteins. Sensitive diagnosis and suppression of avian influenza will help maintain a stable and healthy livestock industry, as well as protect human health.

Development of a receptor-based inhibitory penta-unit-conjugated peptide to enhance anthrax toxin neutralization.

Anthrax toxin is a key virulence factor for Bacillus anthracis. The cell-binding component of anthrax toxin, protective antigen (PA), mediates the entry of the toxin into cells by first binding to the extracellular von Willebrand factor A (VWA) domain of the cellular anthrax toxin receptor (ATR). Herein, we targeted the VWA domain of the cellular receptor to develop a more effective antitoxin agent for neutralization of anthrax toxin. We selected ATR-binding peptides by using a phage display: among these, we identified two novel peptides binding to the ATR with high affinity and specificity, and that neutralized anthrax toxicity in cells. Furthermore, to enhance the functional efficiency of the probes, the peptides were modified and conjugated to three polyvalent probe backbones: a 17 amino-acid-based cyclic form penta-unit, poly-d-lysine (PDL), or the M13 bacteriophage. One of the functionally modified polyvalent peptide probes, the penta-unit-conjugated probe (PUCP) produced the most potent neutralization of anthrax toxin, with half-maximal inhibitory concentration (IC50) of 20 nM. The PUCP disrupted anthrax toxin binding to its receptor and reduced endocytosis of anthrax toxin. This peptide-based approach may, therefore, represent a promising strategy to combat anthrax toxicosis and other bacterial diseases and may be efficient for disease treatment.

Advances in dermatology using DNA aptamer "Aptamin C" innovation: Oxidative stress prevention and effect maximization of vitamin C through antioxidation.

BACKGROUND: Vitamin C (also known as L-ascorbic acid) plays a critical role in reactive oxygen species (ROS) reduction and cell regeneration by protecting cell from oxidative stress. Although vitamin C is widely used in cosmetic and therapeutic markets, there is considerable evidence that vitamin C easily undergoes oxidation by air, pH, temperature, and UV light upon storage. This deficiency of vitamin C decreases its potency as an antioxidant and reduces the shelf-life of products containing vitamin C as its ingredient. To overcome the deficiency of vitamin C, we have developed Aptamin C, an innovative DNA aptamer maximizing the antioxidant efficacy of vitamin C by binding to the reduced form of vitamin C and delaying its oxidation. METHODS: Binding of Aptamin C with vitamin C was determined using ITC analysis. ITC experiment was performed 0.2 mmol/L vitamin C that was injected 25 times in 2 µL aliquots into the 1.8 mL sample cell containing the Aptamin C at a concentration of 0.02 mmol/L. The data were fitted to a one-site binding isotherm using with origin program for ITC v.5.0. RESULTS: To investigate the effect of Aptamin C and vitamin C complex in human skins, both in vitro and clinical tests were performed. We observed that the complex of Aptamin C and vitamin C was significantly effective in wrinkle improvement, whitening effect, and hydration increase. In the clinical test, subjects treated with the complex showed dramatic improvement in skin irritation and itching. No adverse reaction was presented by Aptamin C complex in the test. CONCLUSION: Taken together, these results showed that Aptamin C, an innovative novel compound, should potentially be served as a key cosmeceutical ingredient for a range of skin conditions.

Novel Peptide-Based Inhibitors for Microtubule Polymerization in Phytophthora capsici.

The plant disease Phytophthora blight, caused by the oomycete pathogen Phytophthora capsici, is responsible for major economic losses in pepper production. Microtubules have been an attractive target for many antifungal agents as they are involved in key cellular events such as cell proliferation, signaling, and migration in eukaryotic cells. In order to design a novel biocompatible inhibitor, we screened and identified inhibitory peptides against alpha- and beta-tubulin of P. capsici using a phage display method. The identified peptides displayed a higher binding affinity (nanomolar range) and improved specificity toward P. capsici alpha- and beta-tubulin in comparison to Homo sapiens tubulin as evaluated by fluorometric analysis. One peptide demonstrated the high inhibitory effect on microtubule formation with a nanomolar range of IC50 values, which were much lower than a well-known chemical inhibitor-benomyl (IC50 = 500 µM). Based on these results, this peptide can be employed to further develop promising candidates for novel antifungal agents against Phytophthora blight.

Ultrasensitive Fluorescence Detection of Alzheimer's Disease Based on Polyvalent Directed Peptide Polymer Coupled to a Nanoporous ZnO Nanoplatform.

Amyloid-beta 42 (Aß42), the key biomarker of Alzheimer's disease (AD), aggregates to form neurotoxic amyloid plaques. In this work, we modified two fluorescein isothiocyanate-labeled Aß42-targeting peptides and designed an Aß42-specific ultrasensitive polyvalent-directed peptide polymer (PDPP) to enhance AD diagnosis sensitivity. The dissociation constant of Aß42 by PDPP was 103-fold higher than the single-site-directed peptide. The improved binding was due to the ability of PDPP to detect multiple receptors on the target. The power of the PDPP diagnostic probe was verified in its application to detect Aß42 in cerebrospinal fluid (CSF), which showed a lower limit of detection (LOD) in the fg mL-1 range that is more sensitive than detection by antibodies or single peptides. In addition, we present a novel ultrasensitive diagnostic system using an array of nanoporous ZnO nanoparticles, which play a role in fluorescence signal amplification, to further improve AD diagnosis sensitivity. We enhanced the signal on the basis of the properties of nanoporous ZnO nanoparticles and measured and quantified an ultralow concentration (ag mL-1 range) of Aß42. This PDPP coupled to the nanoporous ZnO-based system is a novel approach to AD diagnosis that might also be useful for the detection of other target biomarkers and clinical applications.

Detection of Nonylphenol with a Gold-Nanoparticle-Based Small-Molecule Sensing System Using an ssDNA Aptamer.

Endocrine-disrupting chemicals (EDCs) threaten many kinds of life throughout the world. These compounds function the same as sexual hormones, inducing precocious puberty, gynecomastia, etc., in the human body. To prevent excess exposure to nonylphenol (NP), a simple and rapid detection system is needed. In this study, we develop a nonylphenol-specific aptamer from a random single-stranded DNA library and test a rapid sensor system based on the aptamer and gold nanoparticles (AuNPs). The aptamer was screened by a methodology involving reduced graphene oxide (rGO). As a result of screening and sequencing, a DNA aptamer was developed that recognizes the target with high binding affinity (Kd = 194.2 ± 65.9 nM) and specificity. The sensor system developed using the aptamer and gold nanoparticles is sensitive (LOD = 2.239 nM). Circular dichroism (CD) spectrometry results show that the free aptamer binds to the target molecule. The aptamer was characterized using gold nanoparticles to measure UV absorbance. Our results suggest that the sensor system developed using this aptamer is useful for field diagnosis of small molecules.

Development of a ssDNA aptamer system with reduced graphene oxide (rGO) to detect nonylphenol ethoxylate in domestic detergent.

Endocrine-disrupting chemicals are a major public health problem throughout the world. In the human body, these compounds functionalize the same as sexual hormones, inducing precocious puberty, gynecomastia, etc. To help prevent this occurrence, a simple detection system is needed. In this study, a nonylphenol ethoxylate (NPE)-specific aptamer was selected by reduced graphene oxide-systematic evolution of ligands by exponential enrichment. A random ssDNA library was incubated with rGO for adsorption, followed by elution with the target molecule. As a result of screening, a DNA aptamer was found that specifically bounds to the target with high binding affinity (Kd  = 100.9 ± 13.2 nM) and had a low limit of detection (LOD = 696 pM). Furthermore, this NPE-binding aptamer bounds selectively to the target. Characterization of the aptamer was confirmed by measuring the fluorescence signal recovery from rGO. In addition, detection of NPE was performed with several water samples, and the detection accuracy was 100 ± 10%. From these results, we expect that this aptamer could be applied to an on-site detection system for NPE in industrial sites or domestic fields.

Optical Sensing Properties of ZnO Nanoparticles Prepared by Spray Pyrolysis.

We studied the optical sensing properties of ZnO nanoparticles prepared by spray pyrolysis. To investigate their optical sensing performance, we incubated peptides on ZnO nanoparticles. The photoluminescence (PL) peak intensity of peptides on the ZnO nanoparticles was higher than that of peptides on the ZnO film or on the glass plate. This observed PL enhancement is attributed to the optical confinement of ZnO nanoparticles. The low-temperature spectra displayed a strong exciton emission peak with multiple sidebands, attributed to the bound exciton and its longitudinal optical phonon sidebands. The strong exciton emission is thought to be the combined effect of optical confinement due to the nanoparticle geometry, reduction of defect emission by thermal annealing, and reduction of non-radiative relaxation at low temperatures.

Development of quantum dot aptasensor and its portable analyzer for the detection of di-2-ethylhexyl phthalate.

We have developed a quantum dot aptasensor (QD-aptasensor) and its accompanying portable analyzer for the detection of di-2-ethylhexyl phthalate (DEHP). This sensor is based on a newly screened aptamer (60-mer) via SELEX and shows a binding affinity of 213 nmol/L with DEHP. The 60-mer aptamer together with its three shorter truncated aptamers (45, 28, and 22-mer) as well as three different DNA probes (12, 9, and 13-mer) were further investigated to form the best combination for the QD-aptasensor. Using a 22-mer-truncated aptamer and a 12-mer DNA probe combination, the QD-aptasensor demonstrated excellent DEHP sensitivity with an LOQ = 0.5 pg/mL as well as good selectivity in the presence of other phthalate analogs. The binding between the truncated aptamers and DEHP was also characterized. Finally, a QD-aptasensor-based portable analyzer was also developed, and its equivalence to the laboratory protocol was established with a correlation coefficient r = 0.86 for DEHP concentrations ranging from 0.0005 to 100 ng/mL.

Inhibition of anthrax lethal factor by ssDNA aptamers.

Anthrax is caused by Bacillus anthracis, a bacterium that is able to secrete the toxins protective antigen, edema factor and lethal factor. Due to the high level of secretion from the bacteria and its severe virulence, lethal factor (LF) has been sought as a biomarker for detecting bacterial infection and as an effective target to neutralize toxicity. In this study, we found three aptamers, and binding affinity was determined by fluorescently labeled aptamers. One of the aptamers exhibited high affinity, with a Kd value of 11.0 ±â€¯2.7 nM, along with low cross reactivity relative to bovine serum albumin and protective antigen. The therapeutic functionality of the aptamer was examined by assessing the inhibition of LF protease activity against a mitogen-activated protein kinase kinase. The aptamer appears to be an effective inhibitor of LF with an IC50 value of 15 ±â€¯1.5 µM and approximately 85% cell viability, suggesting that this aptamer provides a potential clue for not only development of a sensitive diagnostic device of B. anthracis infection but also the design of novel inhibitors of LF.

Development of a ssDNA aptamer for detection of residual benzylpenicillin.

Antibiotics are useful for improving the living conditions of livestock. However, residual antibiotics induce several human diseases such as food-borne illness and infection of carbapenem-resistant Enterobacteriaceae (CRE). In this study, the identification of a benzylpenicillin-specific aptamer was selected by rGO-SELEX (reduced Graphene Oxide-Systematic Evolution of Ligands by EXponential enrichment). A random ssDNA library was incubated with rGO for adsorption and eluted with benzylpenicillin. As a result of the selection process, a DNA aptamer was found that specifically bound to benzylpenicillin with high binding affinity, Kd = 383.4 nM, and had a low limit of detection (LOD) of 9.2 nM. The characterization of the aptamer was performed through the fluorescence recovery signal from rGO surface. In addition, detection of benzylpenicillin was performed in pretreated milk samples, and its detection accuracy was shown to be 100± 10%. This represented that BBA1 was used for fluorescence aptasensor system in real sample. Furthermore, this benzylpenicillin binding aptamer showed high specificity against other antibiotics except for ampicillin. With these advantageous characteristics, we expect that this aptamer could be applied to an on-site detection system for residual benzylpenicillin.

Mutation analysis of the interactions between Mycobacterium tuberculosis caseinolytic protease C1 (ClpC1) and ecumicin.

Ecumicin is a well-known and potent inhibitor of Mycobacterium tuberculosis. Although the target of ecumicin is caseinolytic protease C1 (ClpC1), the exact mechanism by which ecumicin inhibits ClpC1 has not been identified. To analyze ecumicin's action on ClpC1, site-directed mutagenesis was performed on its binding site. The estimated binding residues within ClpC1 to ecumicin were selected via in silico analysis using molecular docking. The selected residues were mutated by site-directed mutagenesis and the effects on ecumicin binding were analyzed. Mutation at the R83 residue, especially the R83A mutation, in ClpC1 resulted in strong resistance to ATPase activation and inhibition of proteolytic activity. In addition, binding of ecumicin to the R83A ClpC1 N-terminal domain (residues 1-145) was not observed in native gel analysis. These results reveal that the R83 residue plays an important role in the binding of ecumicin. This result provides a basis for the development of an anti-tuberculosis agent based on ecumicin derivatives.

Ultra-sensitive detection of kanamycin for food safety using a reduced graphene oxide-based fluorescent aptasensor.

Overuse of antibiotics has caused serious problems, such as appearance of super bacteria, whose accumulation in the human body through the food chain is a concern. Kanamycin is a common antibiotic used to treat diverse infections; however, residual kanamycin can cause many side effects in humans. Thus, development of an ultra-sensitive, precise, and simple detection system for residual kanamycin in food products is urgently needed for food safety. In this study, we identified kanamycin-binding aptamers via a new screening method, and truncated variants were analyzed for optimization of the minimal sequence required for target binding. We found various aptamers with high binding affinity from 34.7 to 669 nanomolar Kdapp values with good specificity against kanamycin. Furthermore, we developed a reduced graphene oxide (RGO)-based fluorescent aptasensor for kanamycin detection. In this system, kanamycin was detected at a concentration as low as 1 pM (582.6 fg/mL). In addition, this method could detect kanamycin accurately in kanamycin-spiked blood serum and milk samples. Consequently, this simple, rapid, and sensitive kanamycin detection system with newly structural and functional analysis aptamer exhibits outstanding detection compared to previous methods and provides a new possibility for point of care testing and food safety.

Development of a novel imaging agent using peptide-coated gold nanoparticles toward brain glioma stem cell marker CD133.

CD133 is known as biomarker for glioblastoma (GBM) and also serves as a marker for cancer stem cells (CSCs), which carry out tumorigenesis and resist conventional therapeutics. The presence of CD133-presenting CSC is a one of the factors in maintenance of the tumorigenic potential of GBM. Thus, CD133 is a potential target for accurate diagnosis of GBM, which could improve its poor prognosis for patients when CSCs are present. Herein we designed a small peptide-based imaging agent with stimulus-responsive properties. A novel small peptide, CBP4, was screened by a phage display technique, and demonstrated binding to the target CD133 (ECD) comparable to that of an antibody. As a quencher, we used gold nanoparticles (GNPs); the targeting peptide was conjugated to GNPs with high efficiency. By means of a quenching effect, the peptide-coated GNP showed 'signal on-off' properties depending upon the presence of the target. In addition, the particles exhibited biocompatibility when localized in the cytosol. Thus, this study demonstrated that the peptide-coated GNPs can be utilized as an imaging agent for accurate diagnosis of GBM, and further as a drug carrier for therapeutic approaches. STATEMENT OF SIGNIFICANCE: The diagnosis and determination of prognosis made by cancer stem cell markers could be a key strategy to eradicate cancer stem cells and cure the cancer. The significance of this study is the characterization of quenching-based signal on-off mechanism and showed that the active targeting via peptide can contribute to the design of a stimulus-responsive cellular imaging agent. Moreover, small peptide based nano complexation showed specific recognition of the target stem cell and internalized on cellular cyotosol with stimulus responsive fluorescence. With its novel biocompatibility, the strategy might be a promising tool for drug carrier systems able to measure and visualize the delivered efficiency at intracellular sites.

Development of potent chemical antituberculosis agents targeting Mycobacterium tuberculosis acetohydroxyacid synthase.

Mycobacterium tuberculosis acetohydroxyacid synthase (MTB-AHAS) has been suggested as a crucial target for antibacterial agents. High-throughput screening of a chemical library was performed to identify potent new inhibitors of MTB-AHAS. Among the 6800 tested compounds, 15 were identified as potent inhibitors, exhibiting >80-90% inhibition of in vitro MTB-AHAS activity at a fixed concentration of 20 µM. Five compounds belonging to the triazolopyrimidine structural class showed greater inhibition potency, with a half-maximum inhibition concentration (IC50 value) in the low micromolar range (0.4-1.24 µM). Furthermore, potent inhibitors demonstrated non-competitive, uncompetitive or mixed-competitive inhibition. Molecular docking experiments with these potent chemicals using a homology model of MTB-AHAS indicated hydrophobic and hydrogen bond interactions with some key herbicide binding site residues with binding energies (ΔG) of -8.04 to -10.68 Kcal/mol, respectively. The binding modes were consistent with inhibition mechanisms, as the chemicals were oriented outside the active site. Importantly, these potent inhibitors demonstrated significant growth inhibition of various clinically isolated multidrug-resistant and extensively drug-resistant M. tuberculosis strains, with 50% minimum inhibitory concentrations (MIC50 values) ranging from 0.2 µg/mL to 0.8 µg/mL, which resemble the MICs of conventional drugs for tuberculosis (isoniazid, 0.1 µg/mL; rifampicin, 0.4 µg/mL). Thus, the identified potent inhibitors show potential as scaffolds for further in vivo studies and might provide an impetus for the development of strong antituberculosis agents targeting MTB-AHAS.

Neural stem cells injured by oxidative stress can be rejuvenated by GV1001, a novel peptide, through scavenging free radicals and enhancing survival signals.

Oxidative stress is a well-known pathogenic mechanism of a diverse array of neurological diseases, and thus, numerous studies have attempted to identify antioxidants that prevent neuronal cell death. GV1001 is a 16-amino-acid peptide derived from human telomerase reverse transcriptase (hTERT). Considering that hTERT has a strong antioxidant effect, whether GV1001 also has an antioxidant effect is a question of interest. In the present study, we aimed to investigate the effects of GV1001 against oxidative stress in neural stem cells (NSCs). Primary culture NSCs were treated with different concentrations of GV1001 and/or hydrogen peroxide (H2O2) for various time durations. The H2O2 decreased the viability of the NSCs in a concentration-dependent manner, with 200µM H2O2 significantly decreasing both proliferation and migration. However, treatment with GV1001 rescued the viability, proliferation and migration of H2O2-injured NSCs. Consistently, free radical levels were increased in rat NSCs treated with H2O2, while co-treatment with GV1001 significantly reduced these levels, especially the intracellular levels. In addition, GV1001 restored the expression of survival-related proteins and reduced the expression of death-associated ones in NSCs treated with H2O2. In conclusion, GV1001 has antioxidant and neuroprotective effects in NSCs following treatment with H2O2, which appear to be mediated by scavenging free radicals, increasing survival signals and decreasing death signals.

Use of Multiple Peptide-Based SERS Probes Binding to Different Epitopes on a Protein Biomarker To Improve Detection Sensitivity.

We propose an analytical strategy to improve the sensitivity for detecting a protein biomarker through signal multiplication by manipulating multiple peptide-based surface-enhanced Raman scattering (SERS) probes to bind the biomarker. Protective antigen (PA) was used as an Anthrax biomarker in this study. For this purpose, five small peptides selective to various PA epitopes with different binding affinities were chosen and peptide-conjugated Au nanoparticle (AuNP) SERS probes were individually prepared using each peptide. Initially, five different SERS probes were separately used to detect PA and the sensitivities were compared. Next, the possibility of enhancing sensitivity by employing multiple SERS probes was examined. Rather than applying the probes simultaneously, which would induce competitive binding, each probe was added sequentially and an optimal probe-addition sequence was determined to provide maximal sensitivity. Finally, PA samples at seven different concentrations were measured with the optimal sequence. The limit of detection (LOD) was 0.1 aM, and the enhancement was more effective at lower PA concentrations. The proposed scheme can be further applicable to detect other protein biomarkers to diagnose various diseases.