Identification of Compounds that Selectively Stabilize Specific G‐Quadruplex Structures by Using a Thioflavin T‐Displacement Assay as a Tool

Small molecules are used in the G‐quadruplex (G4) research field in vivo and in vitro, and there are increasing demands for ligands that selectively stabilize different G4 structures. Thioflavin T (ThT) emits an enhanced fluorescence signal when binding to G4 structures. Herein, we show that ThT can be competitively displaced by the binding of small molecules to G4 structures and develop a ThT‐displacement high‐throughput screening assay to find novel and selective G4‐binding compounds. We screened approximately 28 000 compounds by using three different G4 structures and identified eight novel G4 binders. Analysis of the structural conformation and stability of the G4 structures in presence of these compounds demonstrated that the four compounds enhance the thermal stabilization of the structures without affecting their structural conformation. In addition, all four compounds also increased the G4‐structure block of DNA synthesis by Taq DNA polymerase. Also, two of these compounds showed selectivity between certain Schizosaccharomyces pombe G4 structures, thus suggesting that these compounds or their analogues can be used as selective tools for G4 DNA studies.     

Benzothiazole‐Based Neutral Ratiometric Fluorescence Sensor for Amyloid Fibrils

Early detection of amyloid fibrils is very important for the timely diagnosis of several neurological diseases. Thioflavin‐T (ThT) is a gold standard fluorescent probe for amyloid fibrils and has been used for the last few decades. However, due to its positive charge, ThT is incapable of crossing the blood–brain barrier and cannot be used for in vivo imaging of fibrils. In the present work, we synthesized a neutral ThT derivative, 2‐[2’‐Me,4’‐(dimethylamino)phenyl]benzothiazole (2Me‐DABT), which showed a strong affinity towards the amyloid fibrils. On association with the amyloid fibrils, 2Me‐DABT not only showed a large increase in its emission intensity, but also, unlike ThT, a large blueshift in its emission spectrum was observed. Thus, unlike ThT, 2Me‐DABT is a potential candidate for the ratiometric sensor of the amyloid fibrils. Detailed photophysical properties of 2Me‐DABT in amyloid fibrils and different solvent media were studied to understand its sensory activity. Fluorescence resonance energy transfer (FRET) studies suggested that the sites of localization for ThT and 2Me‐DABT in amyloid fibrils are not same and their average distance of separation in amyloid fibrils was determined. The experimental data was nicely supported by molecular docking studies, which confirmed the binding of 2Me‐DABT in the inner core of the amyloid fibrils.     

Torsional Dynamics of Thioflavin T in Room‐Temperature Ionic Liquids: An Effect of Heterogeneity of the Medium

The ultrafast excited‐state dynamics of a fibril binding dye, thioflavin T (ThT), has been studied in two room‐temperature ionic liquids (RTILs): 1‐Butyl‐1‐methylpyrrolidinium bis(trifluoromethylsulfonyl)imide and methyltrioctylammonium bis(trifluoromethylsulfonyl)imide. Previously, in most studies, it was observed that the excited‐state dynamics of the ThT dye were dependent on the viscosity of the medium. In our study, by using RTILs, we have demonstrated that the excited‐state dynamics of ThT are not only viscosity dependent, but also dependent on the heterogeneous nature of the medium. The effect of structural heterogeneity present in neat RTILs on the excited‐state dynamics of ThT was observed. For both RTILs, excitation wavelength dependency on the emission properties of ThT was observed.     

Binding Modes of Thioflavin T on the Surface of Amyloid Fibrils Studied by NMR

The mechanism for the interaction of thioflavin T (ThT) with amyloid fibrils at the molecular level is not known. Here, we used ¹H NMR spectroscopy to determine the binding mode of ThT on the surface of fibrils from lysozyme and insulin. Relayed rotating‐frame Overhauser enhancements in ThT were observed, indicating that the orientation of ThT is orthogonal to the fibril surface. Importantly, the assembly state of ThT on both surfaces is different. On the surface of insulin fibrils, ThT is oligomeric, as indicated by rapid ¹H spin‐lattice relaxation rate in the rotating frame (R_1ρ), presumably due to intermolecular dipole–dipole interactions between ThT molecules. In contrast, ThT on the surface of lysozyme fibrils is a monomer, as indicated by slower ¹H R_1ρ. These results shed new light into the mechanism for the enhancement of ThT fluorescence and may lead to more efficient detectors of amyloid assemblies, which have escaped detection by ThT in monomer form.     

Programmable Assembly of Nano‐architectures through Designing Anisotropic DNA Origami Patches

Programmable assembly of nanoparticles (NPs) into well‐defined architectures has attracted attention because of tailored properties resulting from coupling effects. However, general and precise approaches to control binding modes between NPs remain a challenge owing to the difficulty in manipulating the accurate positions of the functional patches on the surface of NPs. Here, a strategy is developed to encage spherical NPs into pre‐designed octahedral DNA origami frames (DOFs) through DNA base‐pairings. The DOFs logically define the arrangements of functional patches in three dimensions, owing to the programmability of DNA hybridization, and thus control the binding modes of the caged nanoparticle with designed anisotropy. Applying the node‐and‐spacer approach that was widely used in crystal engineering to design coordination polymers, patchy NPs could be rationally designed with lower symmetry encoded to assemble a series of nano‐architectures with high‐order geometries.     

Identifying the Bond Responsible for the Fluorescence Modulation in an Amyloid Fibril Sensor

An ultrafast intramolecular bond twisting process is known to be the responsible mechanism for the sensing activity of the extensively used amyloid fibril sensor thioflavin T (ThT). However, it is not yet known which one of the two possible single bonds in ThT is actually involved in the twisting process. To resolve this fundamental issue, two derivatives of ThT have been designed and synthesized and subsequently their photophysical properties have been studied in different solvents. It is understood from the present study that the rotation around the central C? C single bond, and not that around the C? N single bond, is primarily responsible for the sensor activity of ThT. Detailed viscosity‐dependent fluorescence studies revealed that the ThT derivative with restricted C? N bond rotation acts as a better sensor than the derivative with free C? N bond rotation. The better sensory activity is directly correlated with a shorter excited‐state lifetime. Results obtained from the photophysical studies of the ThT derivatives have also been supported by the results obtained from quantum chemical calculations.     

ThX – a next-generation probe for the early detection of amyloid aggregates

Neurodegenerative diseases such as Alzheimer''s and Parkinson''s are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, the improved optical properties (extinction coefficient, quantum yield and brightness) of ThX can be used to monitor structural differences in oligomeric species, not observed via traditional ThT imaging.

Introducing ThX, a next-generation ThT derivative that allows for the early detection of amyloid aggregates at the bulk and single-aggregate levels.     

硫黄素T与侧翼连接双链DNA的G-四链体高特异性作用

富G碱基的DNA序列在离子诱导下可形成G-四链体(G4),基于这一构型转化设计了大量的传感检测平台。其中的荧光检测平台是基于G4与荧光小分子的相互作用。但是,G4与荧光小分子的有效结合依赖于G4构型和体系中存在的离子种类和离子浓度,尤其是高Na⁺浓度(140 mmol·L^-1)。那么如何实现G4与荧光小分子普适性地有效结合,并不依赖于体系中的Na⁺和Na⁺浓度,是一个难题。在本研究中,以最简单的富G DNA序列凝血酶适体链TBA (thrombin binding aptamer)为例,在3’端和5’端分别增加10个碱基(TBA-10 bp),K⁺诱导TBA-10 bp形成K⁺稳定TBA (K⁺-TBA,G4)并衔接含有10个互补碱基对的双链DNA (K⁺-TBA-10 bp)。相较于K⁺-TBA,硫磺素T与K⁺-TBA-10 bp结合后的荧光强度增加了100倍,相互作用强度增加了1000倍,而且与体系中的Na⁺ (5-140 mmol·L^-1)无关。结合荧光光谱,紫外吸收光谱和圆二色光谱发现硫磺素T特异性的嵌合于K⁺-TBA和双链DNA衔接处的空腔内。有趣的是,这一结合模式不受G4构型的影响。该研究结果为研究G4与荧光小分子的有效结合提供了新视角,也为拓展G4在生物功能和生化检测领域的应用提供了实验依据。     

Single-molecule investigation of human telomeric G-quadruplex interactions with Thioflavin T

The interactions between human telomere sequence and a typical highly selective G-quadruplex ligand ThT were studied at the single-molecule level through α-hemolysin protein nanopore.     

Guest Binding with Sulfated Cyclodextrins: Does the Size of Cavity Matter?

Sulfated cyclodextrins have recently emerged as potential candidates for producing host–induced guest aggregation with properties better than p-sulfonatocalixarenes that have previously shown numerous applications involving the phenomena of host-induced guest aggregation. In the class of sulfated cyclodextrins (SCD), sulfated β-cyclodextrin (β-SCD) remains the most extensively investigated host molecule. Although it is assumed that the host-induced guest aggregation is predominantly an outcome of interaction of the guest molecule with the charges on the exterior of SCD cavity, it has not been deciphered whether the variation in the cavity size will make a difference in the efficiency of host-induced guest-aggregation process. In this investigation, we present a systematic study of host–induced guest aggregation of a cationic molecular rotor dye, Thioflavin T (ThT) with three different sulfated cyclodextrin molecules, α-SCD, β-SCD and γ-SCD, which differ in their cavity size, using steady-state emission, ground-state absorption and time-resolved emission measurements. The obtained photophysical properties of ThT, upon interaction with different SCD molecules, indicate that the binding strength of ThT with different SCD molecules correlate with the cavity size of the host molecule, giving rise to the strongest complexation of ThT with the largest host molecule (γ-SCD). The binding affinity of ThT towards different host molecules has been supported by molecular docking calculations. The results obtained are further supported with the temperature and ionic strength dependent studies performed on the host-guest complex. Our results indicate that for host–induced guest aggregation, involving oppositely charged molecules, the size of the cavity also plays a crucial role beside the charge density on the exterior of host cavity.     

Construction,Enzyme Response,and Substrate Capacity of a Hyaluronan–Cyclodextrin Supramolecular Assembly

A supramolecular assembly was constructed with a cationic cyclodextrin (EICD) and native hyaluronan (HA). The cationic carboxylic ester pendants on HA support hyaluronidase (HAase)‐responsive sites and the EICD supports artificial carboxylic esterase responsive sites. Substrate‐binding models were investigated by using environment‐sensitive fluorescence probes 2‐p‐toluidino‐6‐naphthalenesulfoniate sodium (2,6‐TNS) and thioflavin T (ThT). On a HA/EICD assembly, EICD was able to bind an anionic substrate and HA and EICD constructed the cationic substrate binding site together. This assembly could be used as a sequential dual‐substrate carrier.     

硫黄素T对淀粉样β-蛋白质40聚集成核动力学的双重影响

荧光染料硫黄素T常用于淀粉样纤维聚集过程的定性定量检测。虽然有研究表明,某些抑制淀粉样蛋白质聚集的小分子抑制剂会与硫黄素T相互作用,影响其测试结果。但硫黄素T如何影响淀粉样蛋白质的聚集成核动力学尚不清晰。本文以淀粉样β-蛋白质40 (Aβ40)为模型,系统研究了硫黄素T对Aβ40聚集成核的影响。研究发现：硫黄素T能够显著改变Aβ40的聚集成核动力学,且影响程度与硫黄素T的浓度密切相关。即在低浓度硫黄素T存在下,Aβ40成核速率的延迟时间先随着硫黄素T浓度的升高而缩短,后随着硫黄素T浓度的升高延迟时间反而延长。但延伸的速率却随硫黄素T浓度的升高而缓慢增大。另外,硫黄素T基本不会影响Aβ40的二级结构和纤维形态。同时,等温滴定微量热实验结果表明,硫黄素T结合Aβ40之间的主要作用力为疏水相互作用。据此,本研究提出硫黄素T对Aβ40聚集成核动力学的双重影响机理。这些结果有助于进一步了解硫黄素T与淀粉样蛋白质的作用特点,为今后硫黄素T在Aβ40聚集成核动力学实验中的使用提供参考。     

Ultrafast Twisting Dynamics of Thioflavin‐T: Spectroscopy of the Twisted Intramolecular Charge‐Transfer State

Understanding the excited‐state properties of thioflavin‐T (ThT) has been of immense importance, because of its efficient amyloid‐sensing ability related to neurodegenerative disorders. The excited‐state dynamics of ThT is studied by using sub‐pico‐ and nanosecond time‐resolved transient absorption techniques as well as density functional theory (DFT)/time‐dependent DFT calculations. Barrierless twisting around the central C?C bond between two aromatic moieties is the dominant process that contributes to the ultrafast dynamics of the S₁ state. The spectroscopic properties of the intramolecular charge‐transfer state are characterized for the first time. The energetics of the S₀ and S₁ states has also been correlated with the experimentally observed spectroscopic parameters and structural dynamics. A longer‐lived transient state populated with a very low yield has been characterized as the triplet state.     

DNA‐Binding Properties of Amidine‐Substituted Spiropyran Photoswitches

Two amidine‐substituted spiropyran derivatives have been characterized with respect to the DNA‐binding properties over a broad pH interval. The two derivatives differ in the number of positive charges. By varying the pH, the protonation state of the derivatives is also changed, allowing for additional variations in the charge distribution. We show that the closed spiro isomer does not bind for either of the two derivatives, whereas the open merocyanine forms bind both in the protonated and in the nonprotonated state, but with dramatically different binding constants. Flow‐oriented linear dichroism (LD) measurements also show that there are differences in the binding modes between the various forms. We rationalize these differences in terms of structure and charge distribution.     

Electrophoretic behavior of DNA‐methyl‐CpG‐binding domain protein complexes revealed by capillary electrophoreses laser‐induced fluorescence

The free solution electrophoretic behavior of DNA‐protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA‐protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA‐methyl‐CpG‐binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA‐protein complexes. This method is based on the effective separation of free DNA and DNA‐protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide‐coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA‐MBD2b complexes, we found that the values of (μ₀/μ)‐1 were directly proportional to the charge‐to‐mass ratios of formed complexes, where the μ₀ and μ are the mobility of free DNA probe and DNA‐protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge‐to‐mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA‐binding properties of the purified MBD2b protein and help to analyze other DNA‐protein complexes. Additionally, this study enhances the understanding of the influence of the charge‐to‐mass ratios of formed DNA‐protein complexes on their separation and electrophoretic behaviors.     

Polycyclic Azoniahetarenes: Assessing the Binding Parameters of Complexes between Unsubstituted Ligands and G‐Quadruplex DNA

Polycyclic azoniahetarenes were employed to determine the effect of the structure of unsubstituted polyaromatic ligands on their quadruplex‐DNA binding properties. The interactions of three isomeric diazoniadibenzo[b,k]chrysenes ( 4 a – c ), diazoniapentaphene ( 5 ), diazoniaanthra[1,2‐a]anthracene ( 6 ), and tetraazoniapentapheno[6,7‐h]pentaphene ( 3 ) with quadruplex DNA were examined by DNA melting studies (FRET melting) and fluorimetric titrations. In general, penta‐ and hexacyclic azoniahetarenes bind to quadruplex DNA (K_b≈10⁶ M ^?1) even in the absence of additional functional side chains. The binding modes of 4 a – c and 3 were studied in more detail by ligand displacement experiments, isothermal titration calorimetry, and CD and NMR spectroscopy. All experimental data indicate that terminal π stacking of the diazoniachrysenes to the quadruplex is the major binding mode; however, because of different electron distributions of the π systems of each isomer, these ligands align differently in the binding site to achieve ideal binding interactions. It is proposed that tetraazonia ligand 3 binds to the quadruplex by terminal stacking with a small portion of its π system, whereas a significant part of the bulky ligand most likely points outside the quadruplex structure, and is thus partially placed in the grooves. Notably, 3 and the known tetracationic porphyrin TMPyP4 exhibit almost the same binding properties towards quadruplex DNA, with 3 being more selective for quadruplex than for duplex DNA. Overall, studies on azonia‐type hetarenes enable understanding of some parameters that govern the quadruplex‐binding properties of parent ligand systems. Since unsubstituted ligands were employed in this study, complementary and cooperative effects of additional substituents, which may interfere with the ligand properties, were eliminated.     

Tridentate N-donor palladium(II) complexes as efficient coordinating quadruplex DNA binders

Fifteen complexes of palladium, platinum, and copper, featuring five different N‐donor tridentate (terpyridine‐like) ligands, were prepared with the aim of testing their G‐quadruplex–DNA binding properties. The fluorescence resonance energy transfer melting assay indicated a striking positive effect of palladium on G‐quadruplex DNA stabilization compared with platinum and copper, as well as an influence of the structure of the organic ligand. Putative binding modes (noncoordinative π stacking and base coordination) of palladium and platinum complexes were investigated by ESI‐MS and UV/Vis spectroscopy experiments, which all revealed a greater ability of palladium complexes to coordinate DNA bases. In contrast, platinum compounds tend to predominantly bind to quadruplex DNA in their aqua form by noncoordinative interactions. Remarkably, complexes of [Pd(ttpy)] and [Pd(tMebip)] (ttpy=tolylterpyridine, tMebip=2,2′‐(4‐p‐tolylpyridine‐2,6‐diyl)bis(1‐methyl‐1H‐benzo[d]imidazole)) coordinate efficiently G‐quadruplex structures at room temperature in less than 1 h, and are more efficient than their platinum counterparts for inhibiting the growth of cancer cells. Altogether, these results demonstrate that both the affinity for G‐quadruplex DNA and the binding mode of metal complexes can be modulated by modifying either the metal or the organic ligand.     

Influence of Mg2+ and Cd2+ on the interaction between sparfloxacin and calf thymus DNA

Mg(2+) and Cd(2+) have different binding capacity to sparfloxacin, and have different combination modes with calf thymus DNA. Selecting these two different metal ions, the influence of them on the binding constants between SPFX and calf thymus DNA, as well as the related mechanism have been studied by using absorption and fluorescence spectroscopy. The result shows that Cd(2+) has weak binding capacity to SPFX in the SPFX-Cd(2+) binary system, but can decrease the binding between SPFX and DNA obviously in SPFX-DNA-Cd(2+) ternary system. Mg(2+) has strong binding capacity to SPFX. It can increase the binding between SPFX and DNA at concentrations <0.01 mM, and decrease the binding between them at concentrations >0.01 mM. Referring to the different modes of Mg(2+) and Cd(2+) binding to DNA, the mechanism of the influence of metal ions on the binding between SPFX and DNA has been proposed. SPFX can directly bind to DNA by chelating DNA base sites. If a metal ion at certain concentration mainly binds to DNA bases, it can decrease the binding constants between SPFX and DNA through competing with SPFX. While if a metal ion at certain concentration mainly binds to phosphate groups of DNA, it can increase the binding constants by building a bridge between SPFX and DNA. The influence direction of metal ions on the binding between quinolone and DNA relays on their binding ratio of affinity for bases to phosphate groups on DNA. Our result supports Palumbo's conclusion that the binding between SPFX and the phosphate groups is the precondition for the combination between SPFX and DNA, which is stabilized through stacking interactions between the condensed rings of SPFX and DNA bases.     

Programmable Assembly of Nano-architectures through Designing Anisotropic DNA Origami Patches

Programmable assembly of nanoparticles (NPs) into well-defined architectures has attracted attention because of tailored properties resulting from coupling effects. However, general and precise approaches to control binding modes between NPs remain a challenge owing to the difficulty in manipulating the accurate positions of the functional patches on the surface of NPs. Here, a strategy is developed to encage spherical NPs into pre-designed octahedral DNA origami frames (DOFs) through DNA base-pairings. The DOFs logically define the arrangements of functional patches in three dimensions, owing to the programmability of DNA hybridization, and thus control the binding modes of the caged nanoparticle with designed anisotropy. Applying the node-and-spacer approach that was widely used in crystal engineering to design coordination polymers, patchy NPs could be rationally designed with lower symmetry encoded to assemble a series of nano-architectures with high-order geometries.     

DNA脱碱基位点存在下非氢键配体的增强结合研究

众所周知,插入剂的DNA特性结合位点位于DNA碱基对之间,然而这种非共价相互作用对于含脱碱基（AP）位点的DNA来讲还没有引起足够的重视,虽然在生物细胞中总是存在着DNA脱碱基位点。本文以原黄素（proflavine,PF）为例研究了插入剂对DNA中AP位点的结合特性。实验结果表明,相对于插入位点而言,AP位点是PF的优先结合位点,AP位点的本征结合常数比插入结合常数高一个数量级以上。此外,PF的结合使含脱碱基位点DNA的热稳定性明显提高,表明PF在脱碱基位点的结合构像明显不同于插入结合时的分子定向。本文结果将有助于判断小分子的DNA结合方式所决定的药物的生物化学及生物物理效用。