Selective recognition of parallel and anti-parallel thrombin-binding aptamer G-quadruplexes by different fluorescent dyes

G-quadruplexes (G4) have been found increasing potential in applications, such as molecular therapeutics, diagnostics and sensing. Both Thioflavin T (ThT) and N-Methyl mesoporphyrin IX (NMM) become fluorescent in the presence of most G4, but thrombin-binding aptamer (TBA) has been reported as the only exception of the known G4-forming oligonucleotides when ThT is used as a high-throughput assay to identify G4 formation. Here, we investigate the interactions between ThT/NMM and TBA through fluorescence spectroscopy, circular dichroism and molecular docking simulation experiments in the absence or presence of cations. The results display that a large ThT fluorescence enhancement can be observed only when ThT bind to the parallel TBA quadruplex, which is induced to form by ThT in the absence of cations. On the other hand, great promotion in NMM fluorescence can be obtained only in the presence of anti-parallel TBA quadruplex, which is induced to fold by K⁺ or thrombin. The highly selective recognition of TBA quadruplex with different topologies by the two probes may be useful to investigate the interactions between conformation-specific G4 and the associated proteins, and could also be applied in label-free fluorescent sensing of other biomolecules.     

Iso-FRET: an isothermal competition assay to analyze quadruplex formation in vitro

Algorithms have been widely used to predict G-quadruplexes (G4s)-prone sequences. However, an experimental validation of these predictions is generally required. We previously reported a high-throughput technique to evidence G4 formation in vitro called FRET-MC. This method, while convenient and reproducible, has one known weakness: its inability to pin point G4 motifs of low thermal stability. As such quadruplexes may still be biologically relevant if formed at physiological temperature, we wanted to develop an independent assay to overcome this limitation. To this aim, we introduced an isothermal version of the competition assay, called iso-FRET, based on a duplex-quadruplex competition and a well-characterized bis-quinolinium G4 ligand, PhenDC3. G4-forming competitors act as decoys for PhenDC3, lowering its ability to stabilize the G4-forming motif reporter oligonucleotide conjugated to a fluorescence quencher (37Q). The decrease in available G4 ligand concentration restores the ability of 37Q to hybridize to its FAM-labeled short complementary C-rich strand (F22), leading to a decrease in fluorescence signal. In contrast, when no G4-forming competitor is present, PhenDC3 remains available to stabilize the 37Q quadruplex, preventing the formation of the F22 + 37Q complex. Iso-FRET was first applied to a reference panel of 70 sequences, and then used to investigate 23 different viral sequences.     

Specific in situ discrimination of amyloid fibrils versus α-helical fibres by the fluorophore NIAD-4

A wide range of human pathologies, including neurodegenerative diseases and other forms of amyloidosis, are associated with the formation of insoluble fibrillar protein aggregates known as amyloids. To gain insights into this process analytical methods are needed, which give quantitative data on the molecular events that are taking place. The dye Thioflavin T (ThT) is widely used for the spectroscopic determination of amyloid fibril formation. Different binding affinities to amyloids at neutral and acidic pH and the frequently observed poor binding at acidic pH are problematic in the use of the cationic ThT. The uncharged fluorescence probe [[5'-(4-hydroxyphenyl)[2,2'-bithiophen]-5-yl]methylene]-propanedinitrile (NIAD-4) has been recently designed by Swager and coworkers, in order to eliminate some of the limitations of ThT. Here we have used this novel dye for in vitro monitoring of the amyloid formation processes of de novo designed model peptides. Amyloid structures were successfully detected by NIAD-4 at neutral as well as acidic pH and no significant fluorescence was detectable in the presence of α-helical fibres. Thus, NIAD-4 proved to be a valuable alternative to ThT for spectroscopic studies on amyloid structures over a broad pH range.     

Phosphorus-containing dendrimers against α-synuclein fibril formation

The aim of this work was to study the effect of phosphorus-containing dendrimers (generations G3 and G4) on the fibrillation of α-synuclein (ASN). The inhibition of fibril formation (filamentous and aggregates) is a potential therapeutic strategy for neurodegenerative disorders such as Parkinson's and other motor disorder neurodegenerative diseases. The interaction between phosphorus-containing dendrimers and ASN was studied by fluorescence spectroscopy. The decrease in the fluorescence intensity of intrisinic tyrosine was the most marked change in the fluorescence intensity observed upon addition of dendrimers. Furthermore, the effect of dendrimers on ASN fibril formation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by fluorescence assays using the dye thioflavin T (ThT). The results showed that phosphorus-containing dendrimers G3 and G4 inhibited fibril formation, when they were used in the ASN/dendrimer ratios 1:0.1 and 1:0.5. However, the higher concentrations of dendrimers did not show this effect.     

Thioflavin T fluorescence anisotropy: an alternative technique for the study of amyloid aggregation

The process of amyloid polymerisation raises keen interest in particular because of the biomedical impact of this process. A variety of analytical methods have been developed to monitor amyloid formation. Thioflavin T (ThT) is the most commonly used dye for detection of amyloid aggregation. Nevertheless, ThT fluorescence enhancement is strongly dependent of fibril morphology. In this study using the HET-s prion fibril model, we show that amyloid formation can be monitored by measuring ThT fluorescence anisotropy. Kinetic parameters obtained by this method are identical to those determined by CD spectrometry. We propose that ThT anisotropy represent an interesting, simple and alternative technique to analyze the amyloid formation process.     

Effect of amino acid variations in the central region of human serum amyloid A on the amyloidogenic properties

Human serum amyloid A (SAA) is a precursor protein of the amyloid fibrils that are responsible for AA amyloidosis. Of the four human SAA genotypes, SAA1 is most commonly associated with AA amyloidosis. Furthermore, SAA1 has three major isoforms (SAA1.1, 1.3, and 1.5) that differ by single amino acid variations at two sites in their 104-amino acid sequences. In the present study, we examined the effect of amino acid variations in human SAA1 isoforms on the amyloidogenic properties. All SAA1 isoforms adopted α-helix structures at 4 °C, but were unstructured at 37 °C. Heparin-induced amyloid fibril formation of SAA1 was observed at 37 °C, as evidenced by the increased thioflavin T (ThT) fluorescence and β-sheet structure formation. Despite a comparable increase in ThT fluorescence, SAA1 molecules retained their α-helix structures at 4 °C. At both temperatures, no essential differences in ThT fluorescence and secondary structures were observed among the SAA1 isoforms. However, the fibril morphologies appeared to differ; SAA1.1 formed long and curly fibrils, whereas SAA1.3 formed thin and straight fibrils. The peptides corresponding to the central regions of the SAA1 isoforms containing amino acid variations showed distinct amyloidogenicities, reflecting their direct effects on amyloid fibril formation. These findings may provide novel insights into the influence of amino acid variations in human SAA on the pathogenesis of AA amyloidosis.     

PAMAM G4 dendrimers affect the aggregation of α-synuclein

α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinson's disease, and inhibition of fibril formation is a potential therapeutic strategy for these conditions. The aim of the present study was to investigate polyamidoamine (PAMAM) dendrimers (generations 4 and 3.5) as inhibitors of fibril formation in vitro by examining their interaction with ASN intrinsic tyrosine fluorescence. Furthermore, the effect of dendrimers on ASN aggregation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by a fluorescence assays using the dye thioflavin T (ThT). The PAMAM G4 dendrimer caused an increase in tyrosine residue fluorescence, and inhibited fibrillation of ASN; inhibited fibrillation was not observed with PAMAM G3.5 dendrimers.     

Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.     

Fluorescence quantum yield of thioflavin T in rigid isotropic solution and incorporated into the amyloid fibrils

In this work, the fluorescence of thioflavin T (ThT) was studied in a wide range of viscosity and temperature. It was shown that ThT fluorescence quantum yield varies from 0.0001 in water at room temperature to 0.28 in rigid isotropic solution (T/η→0). The deviation of the fluorescence quantum yield from unity in rigid isotropic solution suggests that fluorescence quantum yield depends not only on the ultra-fast oscillation of ThT fragments relative to each other in an excited state as was suggested earlier, but also depends on the molecular configuration in the ground state. This means that the fluorescence quantum yield of the dye incorporated into amyloid fibrils must depend on its conformation, which, in turn, depends on the ThT environment. Therefore, the fluorescence quantum yield of ThT incorporated into amyloid fibrils can differ from that in the rigid isotropic solution. In particular, the fluorescence quantum yield of ThT incorporated into insulin fibrils was determined to be 0.43. Consequently, the ThT fluorescence quantum yield could be used to characterize the peculiarities of the fibrillar structure, which opens some new possibilities in the ThT use for structural characterization of the amyloid fibrils.     

Quadruplex formation as a molecular switch to turn on intrinsically fluorescent nucleotide analogs

Quadruplexes are involved in the regulation of gene expression and are part of telomeres at the ends of chromosomes. In addition, they are useful in therapeutic and biotechnological applications, including nucleic acid diagnostics. In the presence of K⁺ ions, two 15-mer sequences d(GGTTGGTGTGGTTGG) (thrombin binding aptamer) and d(GGGTGGGTGGGTGGG) (G3T) fold into antiparallel and parallel quadruplexes, respectively. In the present study, we measured the fluorescence intensity of one or more 2-aminopurine or 6-methylisoxanthopterin base analogs incorporated at loop-positions of quadruplex forming sequences to develop a detection method for DNA sequences in solution. Before quadruplex formation, the fluorescence is efficiently quenched in all cases. Remarkably, G3T quadruplex formation results in emission of fluorescence equal to that of a free base in all three positions. In the case of thrombin binding aptamer, the emission intensity depends on the location of the fluorescent nucleotides. Circular dichroism studies demonstrate that the modifications do not change the overall secondary structure, whereas thermal unfolding experiments revealed that fluorescent analogs significantly destabilize the quadruplexes. Overall, these studies suggest that quadruplexes containing fluorescent nucleotide analogs are useful tools in the development of novel DNA detection methodologies.     

Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence

Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of beta2-microgobulin (beta2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent beta2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid beta-peptide.     

The DEAH-box RNA helicase RHAU binds an intramolecular RNA G-quadruplex in TERC and associates with telomerase holoenzyme

Guanine-quadruplexes (G4) consist of non-canonical four-stranded helical arrangements of guanine-rich nucleic acid sequences. The bulky and thermodynamically stable features of G4 structures have been shown in many respects to affect normal nucleic acid metabolism. In vivo conversion of G4 structures to single-stranded nucleic acid requires specialized proteins with G4 destabilizing/unwinding activity. RHAU is a human DEAH-box RNA helicase that exhibits G4-RNA binding and resolving activity. In this study, we employed RIP-chip analysis to identify en masse RNAs associated with RHAU in vivo. Approximately 100 RNAs were found to be associated with RHAU and bioinformatics analysis revealed that the majority contained potential G4-forming sequences. Among the most abundant RNAs selectively enriched with RHAU, we identified the human telomerase RNA template TERC as a true target of RHAU. Remarkably, binding of RHAU to TERC depended on the presence of a stable G4 structure in the 5'-region of TERC, both in vivo and in vitro. RHAU was further found to associate with the telomerase holoenzyme via the 5'-region of TERC. Collectively, these results provide the first evidence that intramolecular G4-RNAs serve as physiologically relevant targets for RHAU. Furthermore, our results suggest the existence of alternatively folded forms of TERC in the fully assembled telomerase holoenyzme.     

G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin''s peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.     

The interaction of telomeric DNA and C-myc22 G-quadruplex with 11 natural alkaloids

Telomeric DNA and C-myc22 are DNA G-quadruplex (G4)-forming sequences associated with tumorigenesis. Ligands that can facilitate the formation and increase the stabilization of G4 can halt tumor cell proliferation and have been regarded as potential anti-cancer drugs. In the present study, we have investigated the interaction of 11 natural alkaloids with G4 formed by telomeric DNA and C-myc22 sequences. Our results indicated that sanguinarine (San), palmatine (Pal), and berberine (Beb) of the first series (S1) can induce the formation of G4 as well as increase the stabilization ability. Daurisoline (S2-1), O-methyldauricine (S2-2), O-diacetyldaurisoline (S2-3), daurinoline (S2-4), dauricinoline (S2-5), N,N'-dimethyldauricine iodide (S2-6), and N,N'-dimethyldaurisoline iodide (S2-7) of the second series (S2) showed similar stabilization ability. We found that unsaturated ring C, N(+) positively charged centers, and conjugated aromatic rings are key factors to increase the stabilization ability of S1, and we gave some advice on structure modification to S2 through structure-activity study. Besides, we found San and Pal to be cell cycle blocker in G(1). San was speculated to bind to G4 through intercalation or end stacking.     

Flavone Derivatives as Inhibitors of Insulin Amyloid-Like Fibril Formation

Several natural and synthetic flavone derivatives have been reported to inhibit formation of amyloid fibrils or to remodel existing fibrils. These studies suggest that the numbers and positions of hydroxyl groups on the flavone rings determine their effectiveness as amyloid inhibitors. In many studies the primary method for determining the effectiveness of inhibition is measuring Thioflavin T (ThT) fluorescence. This method demonstrably results in a number of false positives for inhibition. We studied the effects of 265 commercially available flavone derivatives on insulin fibril formation. We enhanced the effectiveness of ThT fluorescence measurements by fitting kinetic curves to obtain halftime of aggregation (t ₅₀). Maximal values of ThT fluorescence varied two fold or more in one third of all cases, but this did not correlate with changes in t ₅₀. Changes in t ₅₀ values were more accurate measures of inhibition of amyloid formation. We showed that without a change in an assay, but just by observing complete kinetic curves it is possible to eliminate numbers of false positive and sometimes even false negative results. Examining the data from all 265 flavones we confirmed previous observations that identified the importance of hydroxyl groups for inhibition. Our evidence suggests the importance of hydroxyl groups at locations 5, 6, 7, and 4’, and the absence of a hydroxyl group at location 3, for inhibiting amyloid formation. However, the main conclusion is that the positions are not additive. The structures and their effects must be thought of in the context of the whole molecule.     

Study on the binding of Thioflavin T to beta-sheet-rich and non-beta-sheet cavities

Amyloid fibril formation plays a role in more than 20 diseases including Alzheimer's disease. In vitro detection of these fibrils is often performed using Thioflavin T (ThT), though the ThT binding mode is largely unknown. In the present study, spectral properties of ThT in binding environments representing beta-sheet-rich and non-beta-sheet cavities were examined. Acetylcholinesterase and gamma-cyclodextrin induced a characteristic ThT fluorescence similar to that with amyloid fibrils, whereas beta-cyclodextrin and the beta-sheet-rich transthyretin did not. The cavities of acetylcholinesterase and gamma-cyclodextrin were of similar diameter and only these cavities could accommodate two ThT ions according to molecular modelling. Binding stoichiometry studies also showed a possible binding of two ThT ions. Thus, the characteristic ThT fluorescence is induced in cavities with a diameter of 8-9A and a length able to accommodate the entire length of the ThT ion. The importance of a cavity diameter capable of binding two ThT ions, among others, indicates that an excimer formation is a plausible mechanism for the characteristic fluorescence. We propose a similar ThT binding mode in amyloid fibrils, where cavities of an appropriate size running parallel to the fibril axis have previously been proposed in several amyloid fibril models.