The 2.5th generation enzymatic sensors based on the construction of quasi-direct electron transfer type NAD(P)-Dependent dehydrogenases.

We introduce a versatile method to convert NAD+ or NADP+ -dependent dehydrogenases into quasi-direct electron transfer (quasi-DET)-type dehydrogenases, by modifying with a mediator on the enzyme surface toward the development of 2.5th generation enzymatic sensors. In this study, we use ß-hydroxybutyrate (BHB) dehydrogenase (BHBDh) from Alcaligenes faecalis (AfBHBDh) as a representative NAD+ or NADP+ -dependent dehydrogenase. BHBDhs are important in ketone monitoring, especially for the diagnosis of diabetic ketoacidosis. We modified AfBHBDh with a thiol-reactive phenazine ethosulfate (trPES). We designed, constructed, and modified mutant BHBDhs harboring cysteine residues within 20 Å from the C4 nicotinamide in NAD+/NADH. Mutants Ser65Cys, Thr96Cys, and Lys106Cys showed indistinguishable catalytic activities from the wild-type enzyme, even after trPES modification. These trPES-modified mutants were immobilized on gold disk electrodes via amine coupling with succinimide-groups of dithiobis (succinimidyl hexanoate) self-assembled monolayers for electrochemical measurements. Considering there is a wide range of BHB concentrations, we exploited the linear regression in log scales. The linear range for the sensors with trPES-modified BHBDh mutants Ser65Cys, Thr96Cys, and Lys106Cys were 0.1-4.0 mM in both buffer solution and artificial interstitial fluid (ISF). They have limits of detection of 0.047 mM for Ser65Cys, 0.15 mM for Thr96Cys, and 0.060 mM for Lys106Cys in buffer solution, and 0.12 mM, 0.089 mM, and 0.044 mM in artificial ISF, respectively. These results indicate that redox mediator modification of NAD(P)-dependent dehydrogenases converts them into quasi-DET-type dehydrogenases, thereby enabling their utilization in 2.5th generation enzymatic sensors, which will facilitate the construction of enzymatic sensors suitable for continuous monitoring systems.

Structure of cytotoxic amyloid oligomers generated during disaggregation.

Amyloidosis is characterized by the abnormal accumulation of amyloid proteins. The causative proteins aggregate from monomers to oligomers and fibrils, among which some intermediate oligomers considered as major toxins. Cytotoxic oligomers are generated not only by aggregation but also via fibril disaggregation. However, little is known about the structural characteristics and generation conditions of cytotoxic oligomers produced during disaggregation. Herein, we summarized the structural commonalities of cytotoxic oligomers formed under various disaggregation conditions, including the addition of heat shock proteins or small compounds. In vitro experimental data demonstrated the presence of high-molecular-weight oligomers (protofibrils or protofilaments) that exhibited a fibrous morphology and ß-sheet structure. Molecular dynamics simulations indicated that the distorted ß-sheet structure contributed to their metastability. The tendency of these cytotoxic oligomers to appear under mild disaggregation conditions, implied formation during the early stages of disaggregation. This review will aid researchers in exploring the characteristics of highly cytotoxic oligomers and developing drugs that target amyloid aggregates.

Rapid and Convenient Single-Chain Variable Fragment-Employed Electrochemical C-Reactive Protein Detection System.

Although IgG-free immunosensors are in high demand owing to ethical concerns, the development of convenient immunosensors that alternatively integrate recombinantly produced antibody fragments, such as single-chain variable fragments (scFvs), remains challenging. The low affinity of antibody fragments, unlike IgG, caused by monovalent binding to targets often leads to decreased sensitivity. We improved the affinity owing to the bivalent effect by fabricating a bivalent antibody-enzyme complex (AEC) composed of two scFvs and a single glucose dehydrogenase, and developed a rapid and convenient scFv-employed electrochemical detection system for the C-reactive protein (CRP), which is a homopentameric protein biomarker of systemic inflammation. The development of a point-of-care testing (POCT) system is highly desirable; however, no scFv-based CRP-POCT immunosensors have been developed. As expected, the bivalent AEC showed higher affinity than the single scFv and contributed to the high sensitivity of CRP detection. The electrochemical CRP detection using scFv-immobilized magnetic beads and the bivalent AEC as capture and detection antibodies, respectively, was achieved in 20 min without washing steps in human serum and the linear range was 1-10 nM with the limit of detection of 2.9 nM, which has potential to meet the criteria required for POCT application in rapidity, convenience, and hand-held detection devices without employing IgGs.

Exploration and Application of DNA-Binding Proteins to Make a Versatile DNA-Protein Covalent-Linking Patch (D-Pclip): The Case of a Biosensing Element.

DNA-protein complexes are attractive components with broad applications in various research fields, such as DNA aptamer-enzyme complexes as biosensing elements. However, noncovalent DNA-protein complexes often decrease detection sensitivity because they are highly susceptible to environmental conditions. In this study, we developed a versatile DNA-protein covalent-linking patch (D-Pclip) for fabricating covalent and stoichiometric DNA-protein complexes. We comprehensively explored the database to determine the DNA-binding ability of the candidates and selected UdgX as the only uracil-DNA glycosylase known to form covalent bonds with DNA via uracil, with a binding efficiency >90%. We integrated a SpyTag/SpyCatcher protein-coupling system into UdgX to create a universal and convenient D-Pclip. The usability of D-Pclip was shown by preparing a stoichiometric model complex of a hemoglobin (Hb)-binding aptamer and glucose oxidase (GOx) by mixing at 4 °C. The prepared aptamer-GOx complexes detected Hb in a dose-dependent manner within the clinically required detection range in buffer and human serum without any washing procedures. D-Pclip covalently connects any uracil-inserted DNA sequence and any SpyCatcher-fused protein stoichiometrically; therefore, it has a high potential for various applications.

Real-time monitoring of the amyloid ß1-42 monomer-to-oligomer channel transition using a lipid bilayer system.

This study describes the observation of the transformation of monomeric amyloid ß1-42 (Aß42) into oligomers in a lipid membrane utilizing a lipid bilayer system for electrophysiological measurement. The relevance of oligomers and protofibrils in Alzheimer's disease (AD) is underscored given their significant neurotoxicity. By closely monitoring the shift of Aß42 from its monomeric state to forming oligomeric channels in phospholipid membranes, we noted that this transformation transpired within a 2-h frame. We manipulated the lipid membrane's constitution with components such as glycerophospholipid, porcine brain total lipid extract, sphingomyelin (SM), and cholesterol (Chol.) to effectively imitate nerve cell membranes. Interesting findings showcased Chol.'s ability to foster stable oligomeric channel formation in the lipid membrane, with SM and GM1 lipids potentially enhancing channel formation as well. Additionally, the study identified the potential of a catechin derivative, epigallocatechin gallate (EGCG), in obstructing oligomerization. With EGCG present in the outer solution of the Aß42-infused membrane, a noteworthy reduction in channel current was observed, suggesting the successful inhibition of oligomerization. This conclusion held true in both, prior and subsequent, stages of oligomerization. Our findings shed light on the toxicity of oligomers, promising invaluable information for future advancements in AD treatment strategies.

Development of a liquid chromatography-based versatile bioanalysis for bevacizumab based on pretreatment combining aptamer affinity purification and centrifugal ultrafiltration concentration.

We report on the development of a versatile and accurate bioanalytical method for bevacizumab using a pretreatment method combining affinity purification with anti-idiotypic DNA aptamers and centrifugal ultrafiltration concentration, followed by liquid chromatography (LC)-fluorescence analysis. An affinity purification method using Sepharose beads as an affinity support removed immunoglobulin G and a large amount of coexisting substances in the serum sample. Purified bevacizumab was separated as a single peak by conventional LC and detected fluorometrically, showing good linearity (R2 = 0.999) in the range of 5-200 µg/mL, sufficient to analyze bevacizumab concentrations in the blood of bevacizumab-treated patients. By combining this purification method with a concentration method using a centrifugal filtration device that inhibits non-specific adsorption of bevacizumab, the quantitative range was reduced by a factor of 10 while showing good linearity (R2 = 0.999) in the 0.5-20 µg/mL range. The developed analytical method is expected to be used not only for general bioanalysis of therapeutic mAbs in clinical settings, but also for next-generation antibody drugs that show drug efficacy at low concentrations and for analysis of trace samples.

Regulation of thrombin activity by ligand-induced topological alteration in a thrombin-binding aptamer.

Thrombin-binding aptamer (TBA), which forms a G-quadruplex (G4) structure with anti-parallel topology, interacts with thrombin to inhibit its enzymatic activity. Here we show that the G4-topology-altering ligand L2H2-2M2EA-6LCO (6LCO) changes the anti-parallel topology of TBA G4 to the parallel topology, thereby abrogating the thrombin-inhibitory activity of TBA. This finding suggests that G4 ligands that alter topology may be promising drug candidates for diseases involving G4-binding proteins.

Development of an Anti-Idiotype Aptamer-Based Electrochemical Sensor for a Humanized Therapeutic Antibody Monitoring.

Therapeutic monoclonal antibodies (mAbs) are currently the most effective medicines for a wide range of diseases. Therefore, it is expected that easy and rapid measurement of mAbs will be required to improve their efficacy. Here, we report an anti-idiotype aptamer-based electrochemical sensor for a humanized therapeutic antibody, bevacizumab, based on square wave voltammetry (SWV). With this measurement procedure, we were able to monitor the target mAb within 30 min by employing the anti-idiotype bivalent aptamer modified with a redox probe. A fabricated bevacizumab sensor achieved detection of bevacizumab from 1-100 nM while eliminating the need for free redox probes in the solution. The feasibility of monitoring biological samples was also demonstrated by detecting bevacizumab in the diluted artificial serum, and the fabricated sensor succeeded in detecting the target covering the physiologically relevant concentration range of bevacizumab. Our sensor contributes to ongoing efforts towards therapeutic mAbs monitoring by investigating their pharmacokinetics and improving their treatment efficacy.

Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System.

The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method.

Identification of novel amyloidosis in dogs: α-S1-casein acquires amyloidogenicity in mammary tumor by overexpression and N-terminal truncation.

Mammary tumor-associated amyloidosis (MTAA) in dogs is characterized by amyloid deposition in the stroma of mammary adenoma or carcinoma; however, the amyloid precursor protein remains unknown. We attempted to identify an amyloid precursor protein and elucidated its etiology by characterizing 5 cases of canine MTAA. Proteomic analyses of amyloid extracts from formalin-fixed paraffin-embedded specimens revealed α-S1-casein (CASA1) as a prime candidate and showed the N-terminal truncation of canine CASA1. Both immunohistochemistry and immunoelectron microscopy showed that amyloid deposits or fibrils in MTAA cases were positive for CASA1. Reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction revealed the complete mRNA sequence encoding CASA1, whose expression was significantly higher in the amyloid-positive group. The recombinant protein of the N-terminal-truncated canine CASA1 and the synthetic peptides derived from canine and human CASA1 formed amyloid-like fibrils in vitro. Structural prediction suggested that the N-terminal region of CASA1 was disordered. Previously, full-length CASA1 was reported to inhibit the amyloidogenesis of other proteins; however, we demonstrated that CASA1 acquires amyloidogenicity via excessive synthesis followed by truncation of its disordered N-terminal region. By identifying a novel in vivo amyloidogenic protein in animals and revealing key mechanistic details of its associated pathology, this study provides valuable insights into the integrated understanding of related proteopathies.

Development of Alkaline Phosphatase-Fused Mouse Prion Protein and Its Application in Toxic Aß Oligomer Detection.

Amyloid ß (Aß) oligomers play a key role in the progression of Alzheimer's disease (AD). Multiple forms of Aß assemblies have been identified by in vitro and in vivo analyses; however, it is uncertain which oligomer is highly neurotoxic. Thus, understanding the pathogenesis of AD by detecting toxic Aß oligomers is crucial. In this study, we report a fusion protein of cellular prion protein (PrPc) and alkaline phosphatase (ALP) from Escherichia coli as a sensing element for toxic Aß oligomers. Since the N-terminus domain of PrPc (residue 23-111) derived from mice is known to bind to toxic Aß oligomers in vitro, we genetically fused PrPc23-111 to ALP. The developed fusion protein, PrP-ALP, retained both the binding ability of PrPc and enzymatic activity of ALP. We showed that PrP-ALP strongly bound to high molecular weight (HMW) oligomers but showed little or no affinity toward monomers. The observation that PrP-ALP neutralized the toxic effect of Aß oligomers indicated an interaction between PrP-ALP and toxic HMW oligomers. Based on ALP activity, we succeeded in detecting Aß oligomers. PrP-ALP may serve as a powerful tool for detecting toxic Aß oligomers that may be related to AD progression.

Structure of lactate oxidase from Enterococcus hirae revealed new aspects of active site loop function: Product-inhibition mechanism and oxygen gatekeeper.

l-Lactate oxidase (LOx) is a flavin mononucleotide (FMN)-dependent triose phosphate isomerase (TIM) barrel fold enzyme that catalyzes the oxidation of l-lactate using oxygen as a primary electron acceptor. Although reductive half-reaction mechanism of LOx has been studied by structure-based kinetic studies, oxidative half-reaction and substrate/product-inhibition mechanisms were yet to be elucidated. In this study, the structure and enzymatic properties of wild-type and mutant LOxs from Enterococcus hirae (EhLOx) were investigated. EhLOx structure showed the common TIM-barrel fold with flexible loop region. Noteworthy observations were that the EhLOx crystal structures prepared by co-crystallization with product, pyruvate, revealed the complex structures with "d-lactate form ligand," which was covalently bonded with a Tyr211 side chain. This observation provided direct evidence to suggest the product-inhibition mode of EhLOx. Moreover, this structure also revealed a flip motion of Met207 side chain, which is located on the flexible loop region as well as Tyr211. Through a saturation mutagenesis study of Met207, one of the mutants Met207Leu showed the drastically decreased oxidase activity but maintained dye-mediated dehydrogenase activity. The structure analysis of EhLOx Met207Leu revealed the absence of flipping in the vicinity of FMN, unlike the wild-type Met207 side chain. Together with the simulation of the oxygen-accessible channel prediction, Met207 may play as an oxygen gatekeeper residue, which contributes oxygen uptake from external enzyme to FMN. Three clades of LOxs are proposed based on the difference of the Met207 position and they have different oxygen migration pathway from external enzyme to active center FMN.

Effects of G-Quadruplex Ligands on the Topology, Stability, and Immunostimulatory Properties of G-Quadruplex-Based CpG Oligodeoxynucleotides.

We previously reported that the formation of guanine-quadruplex (G4) structures provides phosphodiester oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanine (CpG ODNs) with higher nuclease resistance and cellular uptake, thereby increasing their immunostimulation efficiency through TLR9 activation. CpG ODNs forming G4 structures (G4 CpG ODNs) are thus potential vaccine adjuvants against infectious diseases. However, the G4 structure changes topology depending on the surrounding environment. Recently, G4 ligands, which are small molecules that bind to G4 ODNs with high affinity, were reported to improve the stability of G4. In this study, we propose to increase the stability and function of G4 CpG ODNs using G4 ligands. We show the effects of two G4 ligands, named L2H2-6OTD (L2H2) and L2G2-2M2EG-6OTD (L2G2), on the topology, stability, and immunostimulatory properties of a monomeric hybrid-type G4 CpG ODN containing CpG motifs in the central loop, named GD3. We found that L2H2 helps maintain the hybrid G4 topology of GD3, whereas L2G2 induces parallel G4 formation. Both G4 ligands increase the thermodynamic and nuclease stability of GD3. However, only GD3 associated with L2H2 binds efficiently to TLR9 and evokes a higher immune response from mouse macrophage-like RAW264 cells. GD3 associated with L2G2 does not bind efficiently to TLR9 and elicits lower cytokine production. Our results demonstrate that the potential to enhance immunostimulatory properties depends on the ability of G4 ligands to maintain and stabilize the hybrid G4 of GD3. We anticipate that our findings will facilitate the development of more effective G4 CpG ODN-based vaccine adjuvants against infectious diseases.

CpG Methylation Altered the Stability and Structure of the i-Motifs Located in the CpG Islands.

Cytosine methylation within the 5'-C-phosphate-G-3' sequence of nucleotides (called CpG methylation) is a well-known epigenetic modification of genomic DNA that plays an important role in gene expression and development. CpG methylation is likely to be altered in the CpG islands. CpG islands are rich in cytosine, forming a structure called the i-motif via cytosine-cytosine hydrogen bonding. However, little is known about the effect of CpG methylation on the i-motif. In this study, The CpG methylation-induced structural changes on the i-motif was examined by thermal stability, circular dichroism (CD) spectroscopy, and native-polyacrylamide gel electrophoresis (Native-PAGE) evaluation of five i-motif-forming DNAs from four cancer-related genes (VEGF, C-KIT, BCL2, and HRAS). This research shows that CpG methylation increased the transitional pH of several i-motif-forming DNAs and their thermal stability. When examining the effect of CpG methylation on the i-motif in the presence of opposite G4-forming DNAs, CpG methylation influenced the proportion of G4 and i-motif formation. This study showed that CpG methylation altered the stability and structure of the i-motif in CpG islands.

In Vitro Continuous 3 Months Operation of Direct Electron Transfer Type Open Circuit Potential Based Glucose Sensor: Heralding the Next CGM Sensor.

BACKGROUND: While continuous glucose monitoring (CGM) systems allow precise and real-time blood glucose control, current electrochemicalbased CGM technologies inherently harbor enzyme instability issues. The direct electron transfer (DET) type open circuit potential (OCP) based enzyme sensing principle can minimize the catalytic turnover of the enzyme reaction, thereby providing longer-term operational stability in future CGM glucose sensors. METHOD: DET-type OCP based glucose sensors were constructed using gold disk electrodes with glucose dehydrogenase capable of DET which was immobilized using a self-assembled monolayer (SAM). The single enzyme layer prepared on the gold electrode was operated in the presence of glucose, using in vitro buffer solution, continuously for over 3 months with the OCP sensor signal monitored every 10 seconds at 25°C. RESULTS: The DET-type OCP glucose sensor was continuously operated for more than 3 months without a significant decrease of the sensor signal and sensitivity (slope). These results suggest that the DET-type OCP glucose sensor is far more stable than the sensor constructed based on the amperometric principle. The long-term stability of DET-type OCP glucose sensor is attributed to the enzyme's minimized catalytic reaction during the operation, thereby extending the lifetime of enzyme. CONCLUSION: The DET-type OCP glucose sensor can be continuously operated for more than 3 months at 25 °C, in vitro without significant decreases in sensor signal and sensitivity. While the further investigation will be required for in vivo validation, the DET-type OCP glucose sensor is ideal for next generation CGM's, especially in long duration implantable use cases.

An Amine-Reactive Phenazine Ethosulfate (arPES)-A Novel Redox Probe for Electrochemical Aptamer-Based Sensor.

Electrochemical aptamer-based biosensors (E-ABs) are attractive candidates for use in biomarker detection systems due to their sensitivity, rapid response, and design flexibility. There are only several redox probes that were employed previously for this application, and a combination of redox probes affords some advantages in target detection. Thus, it would be advantageous to study new redox probes in an E-AB system. In this study, we report the use of amine-reactive phenazine ethosulfate (arPES) for E-AB through its conjugation to the terminus of thrombin-binding aptamer. The constructed E-AB can detect thrombin by square-wave voltammetry (SWV), showing peak current at -0.15 V vs. Ag/AgCl at pH 7, which differs from redox probes used previously for E-ABs. We also compared the characteristics of PES as a redox probe for E-AB to methylene blue (MB), which is widely used. arPES showed stable signal at physiological pH. Moreover, the pH profile of arPES modified thrombin-binding aptamer revealed the potential application of arPES for a simultaneous multianalyte detection system. This could be achieved using different aptamers with several redox probes in tandem that harbor various electrochemical peak potentials. Our findings present a great opportunity to improve the current standard of biological fluid monitoring using E-AB.

Development of a DNA aptamer that binds to the complementarity-determining region of therapeutic monoclonal antibody and affinity improvement induced by pH-change for sensitive detection.

Therapeutic monoclonal antibodies (mAbs) are successful biomedicines; however, evaluation of their pharmacokinetics and pharmacodynamics demands highly specific discrimination from human immunoglobulin G naturally present in the blood. Here, we developed a novel anti-idiotype aptamer (termed A14#1) with extraordinary specificity against the anti-vascular endothelial growth factor therapeutic mAb, bevacizumab. Structural analysis of the antibody-aptamer complex showed that several bases of A14#1 recognized only the complementarity determining region (CDR) of bevacizumab, thereby contributing to its extraordinary specificity. As the CDR of bevacizumab is predicted to be highly positively charged under mildly acidic conditions and that DNA is negatively charged, the affinity of A14#1 to bevacizumab markedly increased at pH 4.7 (KD = 44 pM) than at pH 7.4 (KD = 12 nM). A14#1-based electrochemical detection method capable of detecting 31 pM of bevacizumab at pH 4.7 was thus developed. A14#1 could be potentially useful for therapeutic drug measurement as a novel ligand of bevacizumab.

Transient potentiometry based d-serine sensor using engineered d-amino acid oxidase showing quasi-direct electron transfer property.

d-Serine biosensing has been extensively reported based on enzyme sensors using flavin adenine dinucleotide (FAD) -dependent d-amino acid oxidase (DAAOx), based on the monitoring of hydrogen peroxide generated by the enzymatic reaction, which is affected by dissolved oxygen concentration in the measurement environment in in vivo use. Here we report a novel sensing principle for d-serine, transient potentiometry based d-serine sensor using engineered DAAOx showing quasi-direct electron transfer (DET) property. DAAOx Gly52Val mutant, revealed to possess dye-mediated dehydrogenase activity using artificial synthetic electron acceptors, while its oxidase activity was negligible. The enzyme was immobilized on electrode and was modified with amine-reactive phenazine ethosulfate, resulted an enzyme electrode showing quasi-DET type response. Although OCP based monitoring took more than several minutes to obtain steady state OCP value, the time dependent OCP change monitoring, transient potentiometry, provided rapid and sensitive sensor signals. While dOCP/dt based monitoring was suitable for sensing with longer than 5 s time resolution with d-serine concentration range between 0.5 mM and 5 mM, dOCP/d t based monitoring is suitable for d-serine monitoring with much shorter time resolution (less than 1 s) with high sensitivity with wider dynamic range (20 µM-30 mM). The maximum dOCP/d t was -39.2 ± 2.0 mV/s1/2, the Km(app) was 1.9 mM, and the lower limit of detection was 20 µM. In addition, d-serine monitoring was also possible in the artificial cerebrospinal fluid. The transient potentiometry based sensing reported in this study will be further utilized to realize miniaturized, continuous, real-time, in vivo sensor for d-serine monitoring.

Light-induced production of isobutanol and 3-methyl-1-butanol by metabolically engineered cyanobacteria.

BACKGROUND: Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803). RESULTS: To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO2) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L-1 and 75 mg L-1, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers. CONCLUSION: A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.

Stabilization of VEGF i-motif structure by CpG methylation.

The intercalated motif (i-motif) is a non-canonical nucleic acid structure formed by intercalated hemi-protonated cytosine base pairs (C-C+) under acidic conditions. The i-motif structure formation is involved in biological processes such as transcription regulation. Therefore, the identification of factors controlling i-motif formation is important in elucidating the cellular functions it controls. We previously reported that the VEGF G-quadruplex structure is stabilized by CpG methylation. In this study, the effect of CpG methylation on the stability of the VEGF i-motif structure was investigated. The VEGF i-motif-forming oligonucleotide contains four cytosines on CpG sites, and three of the four cytosines (C4, C15, and C20) are involved in C-C+ formation in the i-motif structure. Circular dichroism (CD) spectra analysis demonstrated that full CpG methylation increased the pH of mid transition (pHT) of the i-motif structure by 0.1, and the melting temperature (Tm) by 5.1 °C in 25 mM sodium cacodylate buffer at pH 5.0. Moreover, single methylation at C4, C15, and C20 increased Tm by 0.5, 1.7, and 2.0 °C in the buffer, respectively. These results demonstrated that CpG methylation stabilized the VEGF i-motif structure.