FRET templated by G-quadruplex DNA: a specific ternary interaction using an original pair of donor/acceptor partners

G-quadruplex represents a suitable scaffold for FRET (fluorescence resonance energy transfer) since its two external quartets offer two well-defined binding sites for concomitant trapping of donor/acceptor partners. Combining selective G-quadruplex binders (macrocyclic bis(quinacridine) BOQ(1) or monomeric quinacridine MMQ(1), donor) with a highly fluorescent DNA probe (thiazole orange, acceptor), we designed a structure-specific FRET-system based on an unprecedented noncovalent ternary complex. This system could be potentially usable as a signature for quadruplex-DNA conformation in solution, but also might offer a unique means for observing cation and ligand binding influence on quadruplex topology.     

Quantitative Detection of G-Quadruplex DNA in Live Cells Based on Photon Counts and Complex Structure Discrimination

G-quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G-quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE , which showed distinguishable fluorescence lifetime responses between G-quadruplexes and other DNA topologies, and fluorescence quantum yield (Φ_f) enhancement upon G-quadruplex binding. We determined two NBTE -G-quadruplex complex structures with high Φ_f values by NMR spectroscopy. The structures indicated NBTE interacted with G-quadruplexes using three arms through π–π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φ_f values and lifetime response of NBTE upon G-quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G-quadruplex DNA in live cells with NBTE and found G-quadruplex DNA content in cancer cells is 4-fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection.     

Dual-functional gadolinium-based copper(II) probe for selective magnetic resonance imaging and fluorescence sensing

A unique gadolinium complex, Nap-DO3A-Gd, comprising a naphthylamine luminescent moiety, a di-2-picolylamine (DPA) binding chelator, and a 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) moiety has been designed and synthesized as a dual-functional probe for selective magnetic resonance imaging and fluorescent sensing of copper(II) in living cells. Nap-DO3A-Gd exhibited a turn-on manner of relaxivity changes and a fluorescent quenching toward Cu(2+). Through the introduction of naphthalamide into the Gd(3+) contrast agent platform to restrict the coordination ability of the DPA chelator and with Gd(3+) coordinating to the DPA moiety to turn away the interferences of other metal cations from Cu(2+) detection, the probe featured selective relaxivity changes toward Cu(2+) over other metal ions and brought unique Cu(2+)-specific luminescent responses. The probe was water-soluble with the luminescent detection limit established at 6 ppb and was successfully used for luminescence imaging detection of copper(II) in living cells. The results demonstrated the efficiency and advantage of our approach in the development of a dual-modality image.     

A new chemical probe for the detection of the cancer-linked galectin-3

A chemical probe was developed for the detection of the emerging cancer marker galectin-3. The probe contains a benzophenone moiety which covalently attaches itself to the protein upon binding and irradiation. Introduction of a fluorescent label via'click' chemistry allows the labelled proteins to be visualized in a gel. With the probe, selective visualization of galectin-3 in protein mixtures was shown and remarkably even in cell lysates.     

FRET‐based Fluorescent and Colorimetric Probe for Selective Detection of Hg(II) and Cu(II) with Dual‐mode

A dual‐function fluorescence resonance energy transfer (FRET)‐based fluorescent and colorimetric probe was rationally fabricated from an energy donor coumarin moiety and an energy acceptor rhodamine moiety linked by a thiohydrazide arm for selective detection of Hg²⁺ and Cu²⁺. Two distinct mechanisms were used for the selective detection. Results revealed that probe 1 showed high fluorescent selectivity towards Hg²⁺ and evident colorimetric selectivity for Cu²⁺, which was suitable for ‘naked‐eye’ detection.     

An intramolecular charge transfer fluorescent probe: synthesis and selective fluorescent sensing of Ag+

An intramolecular charge transfer (ICT) fluorescent probe, in which the thiourea derivative moiety is linked to the fluorescent 4-(dimethylamino) benzamide, has been designed and synthesized. The ions-selective signaling behaviors of the probe were investigated. Upon the addition of Ag+, an overall emission enhancement of 14-fold was observed. Compound 1 displayed highly selective chelation enhanced fluorescence (CHEF) effect with Ag+ over alkali, alkali earth metal ions and some transition metal ions in aqueous methanol solutions. The prominent selective and efficient fluorescent enhancing behavior could be utilized as a new chemosensing probe for the analysis of Ag+ ion in aqueous environment.     

A far-red fluorescent probe for selective G-quadruplex DNA targeting

The development of rapid and simple approaches for detection of G-quadruplex DNA structures has attracted significant attention to disclose their diverse physiological and pathological functions. Thiazole orange (TO) is a common fluorescence probe used for the detection of G-quadruplexes. However, it still suffers from some common problems like non-selective for G-quadruplex and emission in the lower wavelength region of spectrum, thus hampering its further applications. Probes with turn-on fluorescence in the far-red region are highly sought-after due to minimal auto-fluorescence and cellular damage. In this paper, we described a far-red fluorescent probe (L-1) by introducing an amine group into styrylquinolinium scaffold. The experimental results indicated that L-1 exhibited significant fluorescence enhancement when treated with G-quadruplexes but retained weak fluorescence in the presence of duplex DNAs. In addition, this probe also displayed higher binding affinity for parallel G-quadruplexes. The characteristics of L-1 were further investigated with UV–vis spectrophotometry, fluorescence, circular dichroism, KI quenching, FID assay and molecular docking to validate optical photophysical properties, as well as the selectivity, sensitivity and detailed binding mode toward G-quadruplex DNAs.     

A new prodrug-derived ratiometric fluorescent probe for hypoxia: high selectivity of nitroreductase and imaging in tumor cell

Based on the hypoxia prodrug moiety of p-nitrobenzyl, a selective ratiometric fluorescent sensor (RHP) for the detection of microenvironment hypoxia was designed and synthesized. RHP can be selectively activated by bioreductive enzymes (NTR) and results in an evident blue to green fluorescent emission wavelength change in both solution phases and in cell lines, which might be the first fluorescent ratiometric probe for hypoxia in solid tumors.     

Water soluble extended naphthalene diimides as pH fluorescent sensors and G-quadruplex ligands

Extended naphthalene diimides (NDIs) fused to 1,4-dihydropyrazine-2,3-dione, containing two solubilizing moieties, have been synthesized. Fluorescence spectra of the new NDIs were remarkably affected by pH, as the second deprotonation of the dihydropyrazinedione moiety (pK(a) 6.9) switched off the emission. Binding to a G-quadruplex folded oligonucleotide and stoichiometry were evaluated by FRET melting assay and CD analysis. G-quadruplex binding was strongly enhanced shifting from pH 7.4 to pH 6.0 as a consequence of the dihydropyrazinedione moiety protonation. Cytotoxicity studies using two human telomerase-positive cell lines (HT29 and A549) revealed that the best G-quadruplex ligand was very active against the colon cell line, with an EC(50) of 300 nM.     

Highly sensitive and selective cyanide detection via Cu2+ complex ligand exchange

A new type of fluorescent probe (1) with two triazole groups that are conjugated with a carbazole moiety was synthesized by a Cu(I)-catalyzed alkyne-azide click reaction for the selective and sensitive detection of cyanide via fluorescence enhancement by ligand exchange and metal ion removal.     

The first fluorescent sensor for D-glucarate based on the cooperative action of boronic acid and guanidinium groups

A new fluorescent sensor (1) with a recognition unit consisting of a boronic acid moiety and a guanidinium unit shows selective binding of D-glucarate in aqueous solution.     

咪唑修饰萘酰亚胺与DNA的作用及其细胞毒性

设计合成了咪唑及其烷基化咪唑阳离子基团修饰的萘酰亚胺衍生物。利用紫外-可见吸收光谱、荧光光谱、圆二色谱和荧光共振能量转移等方法研究了它们与小牛胸腺DNA(CT DNA)和G-四链体DNA的相互作用。这些化合物对端粒DNA序列的G-四链体有很高的结合能力(K_α4×10~6 L·mol~(-1)),并能够稳定G-四链体。DNA粘度实验结果表明萘酰亚胺衍生物与CT DNA通过插入作用结合。Autodock分子对接模拟结果表明这些化合物通过疏水作用、静电作用或氢键等方式与人体端粒G-四链体的loop和沟槽部分结合。咪唑阳离子基团修饰的萘酰亚胺衍生物4a–c能够定位于细胞核,对肺癌细胞的细胞毒性要高于咪唑基团修饰的萘酰亚胺衍生物3。化合物4a和4b对肺癌细胞A549的细胞毒性明显高于正常人胚肺成纤维细胞MRC-5,表现出良好的抗癌活性。     

A two‐step strategy to visually identify molecularly imprinted polymers for tagged proteins

A practical and relatively simple method to identify molecularly imprinted polymers capable of binding proteins via the molecular tagging (epitope‐like) approach has been developed. In our two‐step method, we first challenge a previously obtained anti‐tag molecularly imprinted polymer with a small molecule including the said tag of choice (a biotin derivative as shown here or other) connected to a linker bound to a second biotin moiety. An avidin molecule partially decorated with fluorescent labels is then allowed to bind the available biotin derivative associated with the polymer matrix. At the end of this simple process, and after washing off all the low‐affinity binding molecules from the polymer matrix, only suitable molecularly imprinted polymers binding avidin through its previously acquired small molecule tag (or epitope‐like probe, in a general case) will remain fluorescent. For confirmation, we tested the selective performance of the anti‐biotin molecularly imprinted polymer binding it to biotinylated alkaline phosphatase. Residual chemical activity of the enzyme on the molecularly imprinted polymer solid support was observed. In all cases, the corresponding nonimprinted polymer controls were inactive.     

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.     

Label-free fluorescent strategy for sensitive detection of tetracycline based on triple-helix molecular switch and G-quadruplex

In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch (THMS) and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, which consists of a central section with a shortened 8-mer aptamer sequence with high affinity to tetracycline and flanked by two arm segments. G-rich oligonucleotide can specifically bind to thioflavin T (ThT) as a signal transduction probe (STP). In the absence of tetracycline, THMS remains stable, the fluorescence of background is low. By the addition of target tetracycline, the aptamer-target binding results in the formation of a structured aptamer-target complex, which disassembles the THMS and releases the STP. The free STP self-assembles into G-quadruplex and specifically binds to ThT which generates a obvious fluorescence enhancement. Using the triple-helix molecular switch, the developed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of tetracycline ranging from 0.2 to 20.0 nmol/L. The detection limit of tetracycline was determined to be 970.0 pmol/L. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.     

A dual-response naphthofluorescein-based fluorescent probe for multiple-channel imaging of cysteine/homocysteine in living cells

A naphthofluorescein-based fluorescent probe with two independent reaction sites (nitro-2,1,3-benzoxadiazole and acrylate moiety) was developed. Integrating these two reaction sites into a single molecule not only can guarantee the selective detection of Cys/Hcy in an elegant fashion, but also can enable Cys/Hcy detection in a multiple-channel responsive manner.     

色胺修饰竹红菌素及其稀土离子配位聚合物与DNA相互作用研究

利用紫外-可见吸收光谱、荧光光谱、圆二色谱(CD)等方法研究了色胺修饰竹红菌素(DTrpHA)及其稀土离子配位聚合物(Y³⁺-DTrpHA, La³⁺-DTrpHA)与小牛胸腺DNA (CT DNA)和G-四链体22AG的相互作用.结果表明, DTrpHA及其配位聚合物中的色胺基团和竹红菌素基团均参与和双链CT DNA的作用,作用方式主要为沟槽作用.与G-四链体DNA作用后, DTrpHA及其配位聚合物中的色胺基团均具有较大的减色效应(> 45%)和峰位红移(≥ 4 nm),说明色胺基团与G-四链体采用外部堆积作用方式结合;而竹红菌素基团的减色效应相对较小且无明显峰位变化,表明竹红菌素基团采用非特异性作用方式与G-四链体的环区碱基或糖-磷酸骨架结合. G-四链体22AG的构象主要为分子内反平行结构,加入DTrpHA及其配位聚合物对G-四链体22AG的构象影响较小. Y³⁺-DTrpHA比DTrpHA和La³⁺-DTrpHA与G-四链体具有更强的相互作用. Y³⁺-DTrpHA使得CT DNA的熔解温度(T_m)上升了仅1.9 ℃,而使G-四链体的熔解温度上升了13.1 ℃.荧光嵌插剂置换实验 (FID)结果表明, Y³⁺-DTrpHA对G-四链体具有良好亲和性,具有较小的^G4DC₅₀值(使噻唑橙/G-四链体体系荧光下降50%所需配体或配合物的浓度)和较高的G-四链体选择性.     

A fluorescent probe for monitoring nitric oxide production using a novel detection concept

A fluorescent probe using a novel 'spin exchange' concept was developed for monitoring nitric oxide (NO) production. The probe is composed of 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) labeled with acridine and N-dithiocarboxysarcosine (DTCS)-Fe(II) complex. When the non-fluorescent acridine-TEMPO was incubated with DTCS-Fe(II) complex in buffer solution, the nitroxide radical in the acridine-TEMPO interacted with the Fe(II) through a redox interaction. This interaction recovered the fluorescence based on the acridine moiety. The addition of an NO-releasing reagent caused a fluorescent decrease of the probe due to the irreversible binding of NO to the Fe(II), and the amount of the fluorescent decrease strictly corresponded to that of released NO. Using this probe, less than 100 nM of NO can be detected. This probe system is not only useful for monitoring direct production of NO in an aqueous solution, but is also interesting as a basic concept by which to construct new types of NO fluorescent probes.     

Simple and sensitive fluorescence sensor for detection of potassium ion in the presence of high concentration of sodium ion using berberine–G-quadruplex complex as sensing element

In this work, a novel potassium ion (K⁺) sensor is presented using berberine–G-quadruplex complex as a fluorescent probe. This sensor is based on the K⁺that can induce the G-rich DNA to form G-quadruplex conformation. The G-quadruplex can bind berberine to form berberine–G-quadruplex complex, resulting in remarkable enhancement of fluorescence emission of the berberine–G-quadruplex system. In the presence of 800 mM sodium ion (Na⁺), the fluorescence of the berberine–G-quadruplex complex increased linearly with increasing K⁺ concentration in the range of 0.005–1.0 mM. The turn-on fluorescent assay is simple, inexpensive, and highly sensitive. We observed that Na⁺ in 10,000-fold molar excess does not interfere. The molecular mechanisms which produce enhanced fluorescence of berberine were discussed.     

Near‐Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design,Synthesis, and Application in Living Cells and Zebrafish

Fluorescence imaging of tyrosinase (a cancer biomarker) in living organisms is of great importance for biological studies. However, selective detection of tyrosinase remains a great challenge because current fluorescent probes that contain the 4‐hydroxyphenyl moiety show similar fluorescence responses to both tyrosinase and some reactive oxygen species (ROS), thereby suffering from ROS interference. Herein, a new tyrosinase‐recognition 3‐hydroxybenzyloxy moiety, which exhibits distinct fluorescence responses for tyrosinase and ROS, is proposed. Using the recognition moiety, we develop a near‐infrared fluorescence probe for tyrosinase activity, which effectively eliminates the interference from ROS. The high specificity of the probe was demonstrated by imaging and detecting endogenous tyrosinase activity in live cells and zebrafish and further validated by an enzyme‐linked immunosorbent assay. The probe is expected to be useful for the accurate detection of tyrosinase in complex biosystems.