Combinatorial screening for therapeutics in ATTRv amyloidosis identifies naphthoquinone analogues as TTR-selective amyloid disruptors.

Hereditary ATTR amyloidosis is caused by the point mutation in serum protein transthyretin (TTR) that destabilizes its tetrameric structure to dissociate into monomer. The monomers form amyloid fibrils, which are deposited in peripheral nerves and organs, resulting in dysfunction. Therefore, a drug that dissolves amyloid after it has formed, termed amyloid disruptor, is needed as a new therapeutic drug. Here, we first established a high throughput screening system to find TTR interactors from the LOPAC1280 compound library. Among the hit compounds, thioflavin T-based post-treatment assay determined lead compounds for TTR amyloid disruptors, NSC95397 and Gossypol, designated as B and R, respectively. Because these compounds have naphthoquinone-naphthalene structures, we tested 100 naphthoquinone derivatives, and found 10 candidate compounds that disrupted TTR amyloid. Furthermore, to determine whether these 10 compounds are selective for TTR amyloid, we evaluated them against beta-amyloid (Aß1-42). We found two compounds that were selective for TTR and did not disrupt Aß-derived amyloid. Therefore, we succeeded in identifying TTR-selective amyloid disruptors, and demonstrated that naphthoquinone compounds are useful structures as amyloid disruptors. These findings contribute to the on-going efforts to discover new therapeutic tools for TTR amyloidosis.

Investigating Chaperone like Activity of Green Silver Nanoparticles: Possible Implications in Drug Development.

Protein aggregation and amyloidogenesis have been associated with several neurodegenerative disorders like Alzheimer's, Parkinson's etc. Unfortunately, there are still no proper drugs and no effective treatment available. Due to the unique properties of noble metallic nanoparticles, they have been used in diverse fields of biomedicine like drug designing, drug delivery, tumour targeting, bio-sensing, tissue engineering etc. Small-sized silver nanoparticles have been reported to have anti-biotic, anti-cancer and anti-viral activities apart from their cytotoxic effects. The current study was carried out in a carefully designed in-vitro to observe the anti-amyloidogenic and inhibitory effects of biologically synthesized green silver nanoparticles (B-AgNPs) on human serum albumin (HSA) aggregation taken as a model protein. We have used different biophysical assays like thioflavin T (ThT), 8-Anilino-1-naphthalene-sulphonic acid (ANS), Far-UV CD etc. to analyze protein aggregation and aggregation inhibition in vitro. It has been observed that the synthesized fluorescent B-AgNPs showed inhibitory effects on protein aggregation in a concentration-dependent manner reaching a plateau, after which the effect of aggregation inhibition was significantly declined. We also observed meaningful chaperone-like aggregation-inhibition activities of as-synthesized florescent B-AgNPs in astrocytes.

Trypsin Induced Degradation of Amyloid Fibrils.

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.

Fluorescence Lifetime and Intensity of Thioflavin T as Reporters of Different Fibrillation Stages: Insights Obtained from Fluorescence Up-Conversion and Particle Size Distribution Measurements.

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.

Identification of two principal amyloid-driving segments in variable domains of Ig light chains in systemic light-chain amyloidosis.

Systemic light-chain amyloidosis (AL) is a human disease caused by overexpression of monoclonal immunoglobulin light chains that form pathogenic amyloid fibrils. These amyloid fibrils deposit in tissues and cause organ failure. Proteins form amyloid fibrils when they partly or fully unfold and expose segments capable of stacking into ß-sheets that pair and thereby form a tight, dehydrated interface. These structures, termed steric zippers, constitute the spines of amyloid fibrils. Here, using a combination of computational (with ZipperDB and Boston University ALBase), mutational, biochemical, and protein structural analyses, we identified segments within the variable domains of Ig light chains that drive the assembly of amyloid fibrils in AL. We demonstrate that there are at least two such segments and that each one can drive amyloid fibril assembly independently of the other. Our analysis revealed that peptides derived from these segments form steric zippers featuring a typical dry interface with high-surface complementarity and occupy the same spatial location of the Greek-key immunoglobulin fold in both λ and κ variable domains. Of note, some predicted steric-zipper segments did not form amyloid fibrils or assembled into fibrils only when removed from the whole protein. We conclude that steric-zipper propensity must be experimentally validated and that the two segments identified here may represent therapeutic targets. In addition to elucidating the molecular pathogenesis of AL, these findings also provide an experimental approach for identifying segments that drive fibril formation in other amyloid diseases.

Stabilizing proteins to prevent conformational changes required for amyloid fibril formation.

Amyloid fibrillation is associated with several human maladies, such as Alzheimer's, Parkinson's, Huntington's diseases, prions, amyotrophic lateral sclerosis, and type 2 diabetes diseases. Gaining insights into the mechanism of amyloid fibril formation and exploring novel approaches to fibrillation inhibition are crucial for preventing amyloid diseases. Here, we hypothesized that ligands capable of stabilizing the native state of query proteins might prevent protein unfolding, which, in turn, may reduce the propensity of proteins to form amyloid fibrils. We demonstrated the efficient inhibition of amyloid formation of the human serum albumin (HSA) (up to 85%) and human insulin (up to 80%) by a nonsteroidal anti-inflammatory drug, ibuprofen (IBFN). IBFN significantly increases the conformational stability of both HSA and insulin, as confirmed by differential scanning calorimetry (DSC). Moreover, increasing concentration of IBFN boosts its amyloid inhibitory propensity in a linear fashion by influencing the nucleation phase as assayed by thioflavin T fluorescence, transmission electron microscopy, and dynamic light scattering. Furthermore, circular dichroism analysis supported the DSC results, showing that IBFN binds to the native state of proteins and almost completely prevents their tendency to lose secondary and tertiary structures. Cell toxicity assay confirms that species formed in the presence of IBFN are less toxic to neuronal cells (SH-SY5Y). These results demonstrate the feasibility of using a small molecule to stabilize the native state of proteins, thereby preventing the amyloidogenic conformational changes, which appear to be the common link in several human amyloid diseases.

A rapid fluorometric method for determination of aflatoxin B1 in plant-derived food by using a thioflavin T-based aptasensor.

A fluorometric aptamer-based method is described for the determination of aflatoxin B1 (AFB1). The fluorescent dye thioflavin T (ThT) forms a complex with the aptamer against AFB1 (aptamer/ThT), and the fluorescence of the complex is strongly enhanced. On addition of AFB1, it will bind to the aptamer and release ThT. The fluorescence of free ThT is much weaker. The fluorescence of the system, best measured at excitation/emission wavelengths of 440/487 nm, drops gradually in the AFB1 concentration range from 0.2 to 200 ng·mL-1, exhibiting good linearity. The detection limits are 0.2 ng·mL-1 in buffer solution, and 1 ng·mL-1 when applied to plant-derived food. The recovery of AFB1 from spiked foodstuff ranges from 74.7% to 121%. The assay can be performed within 20 min. Graphical abstract Schematic presentation of label-free thioflavin T (ThT)-based fluorescence aptasensor using aflatoxin B1(AFB1) aptamer/ThT G-quadruplex complex and the AFB1 aptamer/AFB1 complex fluorescent signal system for the rapid and sensitive detection of AFB1 in soy sauce, spirits, rice, corn and peanuts.

Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T.

The persistence of high concentrations of beta-2-microglobulin (ß2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of ß2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length ß2M (ß2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6ß2m and ΔN10ß2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of ß2M amyloid fibrils with affinity ~104 M-1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with ß2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-ß2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10ß2m fibrils from other ß2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between ß2m and ΔN6ß2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the ß2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit ß2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

Soluble polymorphic bank vole prion proteins induced by co-expression of quiescin sulfhydryl oxidase in E. coli and their aggregation behaviors.

BACKGROUND: The infectious prion protein (PrPSc or prion) is derived from its cellular form (PrPC) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrPC to PrPSc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrPC (BVPrP) is highly susceptible to PrPSc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease. RESULTS: To explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion. CONCLUSIONS: Our results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.

A Sensitive and Label-Free Pb(II) Fluorescence Sensor Based on a DNAzyme Controlled G-Quadruplex/Thioflavin T Conformation.

Pb(II) can cause serious damaging effects to human health, and thus, the study of Pb2+ detection methods to sensitively and selectively monitor Pb(II) pollution has significant importance. In this work, we have developed a label-free fluorescence sensing strategy based on a Pb(II) DNAzyme cleavage and the ThT/G-quadruplex complex. In the presence of Pb(II), a G-rich tail was cut and released from the substrate strand, which then would form a G-quadruplex structure by combination with ThT dye. The fluorescence signal increase was then measured for sensitive Pb(II) quantification with a limit of detection of 0.06 nM. Our sensor also demonstrated high selectivity against six different metal ions, which is very important for the analysis of complex samples.

A Label-Free Fluorescent Amplification Strategy for High-Sensitive Detection of Pseudomonas aeruginosa based on Protective-EXPAR (p-EXPAR) and Catalytic Hairpin Assembly.

This study presents a fluorescent mechanism for two-step amplification by combining two widely used techniques, exponential amplification reaction (EXPAR) and catalytic hairpin assembly (CHA). Pseudomonas aeruginosa (P. aeruginosa) engaged in competition with the complementary DNA in order to attach to the aptamer that had been fixed on the magnetic beads. The unbound complementary strand in the liquid above was utilized as a trigger sequence to initiate the protective-EXPAR (p-EXPAR) process, resulting in the generation of a substantial quantity of short single-stranded DNA (ssDNA). The amplified ssDNA can initiate the second CHA amplification process, resulting in the generation of many double-stranded DNA (dsDNA) products. The CHA reaction was initiated by the target/trigger DNA, resulting in the release of G-quadruplex sequences. These sequences have the ability to bond with the fluorescent amyloid dye thioflavin T (ThT), generating fluorescence signals. The method employed in this study demonstrated a detection limit of 16 CFU/ml and exhibited a strong linear correlation within the concentration range of 50 CFU/ml to 105 CFU/ml. This method of signal amplification has been effectively utilized to create a fluorescent sensing platform without the need for labels, enabling the detection of P. aeruginosa with high sensitivity.

Engineering of a DNAzyme-Based dimeric G-quadruplex rolling circle amplification for robust analysis of lead ion.

Detecting heavy metal pollution, particularly lead ion (Pb2⁺) contamination, is imperative for safeguarding public health. In this study, we introduced an innovative approach by integrating DNAzyme with rolling circle amplification (RCA) to propose an amplification sensing method termed DNAzyme-based dimeric-G-quadruplex (dimer-G4) RCA. This sensing approach allows for precise and high-fidelity Pb2⁺ detection. Strategically, in the presence of Pb2⁺, the DNAzyme undergoes substrate strand (S-DNA) cleavage, liberating its enzyme strand (E-DNA) to prime isothermal amplification. This initiates the RCA process, producing numerous dimer-G-Quadruplexes (dimer-G4) as the signal reporting transducers. Compared to conventional strategies using monomeric G-quadruplex (mono-G4) as the reporting transducers, these dimer-G4 structures exhibit significantly enhanced fluorescence when bound with Thioflavin T (ThT), offering superior target signaling ability for even detection of Pb2⁺ at low concentration. Conversely, in the absence of Pb2⁺, the DNAzyme structure remains intact so that no primers can be produced to cause the RCA initiation. This nucleic acid amplification-based Pb2⁺ detection method combing with the high specificity of DNAzymes for Pb2⁺ recognition ensures highly sensitive detection of Pb2+ with a detection limit of 0.058 nM, providing a robust tool for food safety analysis and environmental monitoring.

The Use of Thioflavin T for the Estimation and Measurement of the Plasma Membrane Electric Potential Difference in Different Yeast Strains.

The use of the cationic, dye thioflavin T (ThT), to estimate the electric plasma membrane potential difference (PMP) via the fluorescence changes and to obtain its actual values from the accumulation of the dye, considering important correction factors by its binding to the internal components of the cell, was described previously for baker's yeast. However, it was considered important to explore whether the method developed could be applied to other yeast strains. Alternative ways to estimate the PMP by using flow cytometry and a multi-well plate reader are also presented here. The methods were tested with other strains of Saccharomyces cerevisiae (W303-1A and FY833), as well as with non-conventional yeasts: Debaryomyces hansenii, Candida albicans, Meyerozyma guilliermondii, and Rhodotorula mucilaginosa. Results of the estimation of the PMP via the fluorescence changes under different conditions were adequate with all strains. Consistent results were also obtained with several mutants of the main monovalent transporters, validating ThT as a monitor for PMP estimation.

Rapid screening of aptamers for fluorescent targets by integrated digital PCR and flow cytometry.

In this paper, we report the development of a new strategy termed integrated digital PCR-fluorescence activated sorting based SELEX (IFS-SELEX) that enables rapid screening of aptamers against fluorescent targets. Initially, this strategy employs an integrated digital PCR system to amplify each sequence of a preliminarily enriched library, which is obtained by a traditional SELEX method, on the surface of polystyrene beads. Then, the as-prepared beads are incubated with the fluorescent target and then subjected to fluorescence-activated sorting. Since only those sequences with high binding affinity for the target are collected and sequenced, unnecessary analysis of ineligible sequences is avoided by this method, and the aptamer selection process is thereby greatly streamlined. As a proof-of-concept, we applied this strategy for the screening of aptamers against two fluorescent targets, i.e., ciprofloxacin (CFX) and thioflavin T (ThT), and successfully obtained corresponding sequences with low dissociation constants. The binding affinities of aptamers for ThT were well associated with the sorting regions defined in the fluorescence channel of the flow cytometry process. The experimental results demonstrated that the as-designed IFS-SELEX method can serve as a universal platform for rapid, facile, and efficient aptamer selection against various fluorescent targets.

Bioactive Phytocompounds: Anti-amyloidogenic Effects Against Hen Egg-White Lysozyme Aggregation.

Amyloidosis is the process of fibril formation responsible for causing several diseases in the human being that involve protein aggregation such as Alzheimer's, Parkinson's, Huntington's disease, and type II diabetes. Natural phytocompounds such as curcumin shown promising anti-amyloidogenic activity. In the present study, selective phytocompounds such as piperine, cinnamaldehyde, eugenol, and cuminaldehyde present in Piper nigrum L, Cinnamomum zeylanicum Blume, Eugenia caryophyllus Thumb, and Cuminum cyminum L, respectively were analyzed for anti-amyloidogenic activity using hen egg white-lysozyme (HEWL) as a model system. Out of the selected phytocompounds, piperine showed the most significant anti-amyloidogenic activity, as evident from in vitro assays that were validated by in silico molecular docking study. Piperine showed 64.7 ± 3.74% inhibition of amyloid formation at 50 µM concentration, as observed by Thioflavin T assay. Subsequently, the anti-amyloidogenic activity of piperine was further validated by congo red, intrinsic fluorescence assay, and transmission electron microscopy analysis. The in silico molecular binding interaction showed piperine with the highest docking score and glide energy. Piperine was found to be interacting with amyloidogenic region residues and Trp62, the most important residue involved in the amyloidogenesis process. In conclusion, piperine can be used as a positive lead for a potential therapeutic role in targeting diseases involved amyloidogenesis.

Effect of the fluorescent probes ThT and ANS on the mature amyloid fibrils.

Fluorescent probes thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) are widely used to study amyloid fibrils that accumulate in the body of patients with serious diseases, such as Alzheimer's, Parkinson's, prion diseases, etc. However, the possible effect of these probes on amyloid fibrils is not well understood. In this work, we investigated the photophysical characteristics, structure, and morphology of mature amyloid fibrils formed from two model proteins, insulin and lysozyme, in the presence of ThT and ANS. It turned out that ANS affects the secondary structure of amyloids (shown for fibrils formed from insulin and lysozyme) and their fibers clusterization (valid for lysozyme fibrils), while ThT has no such effects. These results confirm the differences in the mechanisms of these dyes interaction with amyloid fibrils. Observed effect of ANS was explained by the electrostatic interactions between the dye molecule and cationic groups of amyloid-forming proteins (unlike hydrophobic binding of ThT) that induce amyloids conformational changes. This interaction leads to weakening repulsion between positive charges of amyloid fibrils and can promote their clusterization. It was shown that when fibrillogenesis conditions and, consequently, fibrils structure is changing, as well as during defragmentation of amyloids by ultrasonication, the influence of ANS to amyloids does not change, which indicates the universality of the detected effects. Based on the obtained results, it was concluded that ANS should be used cautiously for the study of amyloid fibrils, since this fluorescence probe have a direct effect on the object of study.

Ratiometric Fluorescence Sensor for the MicroRNA Determination by Catalyzed Hairpin Assembly.

A novel catalyzed hairpin assembly-based turn-on ratiometric fluorescence biosensor was constructed for the determination of microRNA-122 (miRNA-122) by using 2-aminopurine (2-AP) and thioflavin T (ThT) as detection signal sources. Hairpin DNA sequence (H1) includes the complementary strands of miRNA-122 and G-quadruplex-forming sequence. When miRNA-122 was presented, hybridization occurred between miRNA-122 and part of H1, causing a double-stranded DNA and a G-quadruplex formed. The formed double-stranded DNA significantly decreased the fluorescence intensity of 2-AP. Furthermore, after binding with ThT, the formed G-quadruplex led to the fluorescent enhancement. The hairpin DNA sequence (H2) hybridized with the unfolded H1 and displaced miRNA-122. Finally, the displaced miRNA-122 again hybridized with the H1 and initiated cycle amplification. This sensor showed a linear ranges of 0.5-50 nM and the limit of detection for miRNA-122 assay was 72 pM (with the lowest measured concentration of 500 pM) for determination of miRNA-122 when no other miRNA was present. Measurements on cell lysates from 100, 1000, and 10 000 cells of three different cell lines provided increasing signal ratios, which showed the application potential of the sensor for miRNA determination in real samples.

Deciphering the mechanistic insight into the stoichiometric ratio dependent behavior of Cu(II) on BSA fibrillation.

Various metal ions are recently implicated in protein aggregates and are associated with numerous neurodegenerative diseases. In the present work, we have scrutinized the effect of stoichiometric variation of Cu(II) on BSA fibrillation at physiological pH 7.4 through Thioflavin-T dye binding study, residual protein concentration, Fourier Transform Infrared spectroscopy (FTIR), Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM) and Isothermal Titration Calorimetry (ITC). Fibrillation kinetics was studied through ThT fluorescence, which illustrated dependency on stoichiometric ratio of Cu(II). The ThT intensity data were fitted to a single exponential expression to determine the aggregation rate, k, which revealed a slight lower k value for 1:1 ratio of BSA-Cu(II) reaction as compared to BSA alone whereas k value for 1:2 ratio of BSA-Cu(II) reaction was higher. Higher equilibrium residual concentration in case of 1:1 ratio of BSA-Cu(II) agreed to the lower aggregation rate. IR spectroscopy revealed the presence of increased ß-sheet proportion to the detriment of α-helix conformation with increasing concentration of Cu(II) and illustrated maximum ß-sheet proportion in 1:2 ratio of BSA-Cu(II). These results were combined with scattering results that showed the higher average hydrodynamic radius (Z-average value) of aggregates in 1:2 ratio of BSA-Cu(II) with respect to BSA and 1:1 ratio of BSA-Cu(II). AFM analysis confirmed the fibril formation. ITC analysis has shown the presence of two binding sites with many fold difference in binding affinity at prescribed in vitro conditions. An N-terminal binding site with many fold higher binding affinity was found as the first Cu(II) binding site. The free thiol group of the cysteine residue positioned at 34 (cys-34) in BSA was covalently capped and this modified BSA also showed the presence of two binding sites, which declined the cys-34 site as the second Cu(II) bind site. Therefore Zn(II) binding site was predicted as the second Cu(II) binding site. The binding of Cu(II) at N-terminal binding site marks a square planner geometry; while that at zinc binding site marks a rigid coordination geometry. Conclusively, analyzed data has concluded that the variation in Cu(II) efficacy in the different stoichiometric ratio results because of two different coordination types in different stoichiometric ratios.

Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.

To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design.

Stable label-free fluorescent sensing of biothiols based on ThT direct inducing conformation-specific G-quadruplex.

In this work, a new, label-free, turn-on fluorescent sensor for biothiols detection based on ThT direct inducing conformation-specific G-quadruplex is developed. The sensing approach is based on a conformational switch of oligonucleotide controlled by Hg(2+) and a commercially available water-soluble fluorescent dye, Thioflavin T (ThT). A noticeable fluorescence light-up in ThT on binding to the G-quadruplex grants the sensor excellent sensitivity. The specific quadruplex conformation induced directly by ThT and pronounced structural selectivity of ThT for G-quadruplexes could generate more stable luminescence and make sure high specificity in complex biological samples. The present assay allows for the selective determination of cysteine and glutathione in the range of 2.0 × 10(-8)-2.5 × 10(-6)M and 3.0 × 10(-8)-2.0 × 10(-6)M with a detection limit of 8.4 nM and 13.9 nM respectively. The diagnostic capability and potential in practical applications of this method have been demonstrated by detecting biothiols in human blood serum.