Binding of malachite green promotes stability and shows preference for a human telomere DNA G-quadruplex

A single-stranded human telomere DNA sequence can fold into an intramolecular G-quadruplex structure, which has been shown to inhibit telomerase activity. Small molecules that selectively target and stabilise the G-quadruplex structure have been proposed as potential anticancer drugs. In this study, we analysed the properties of binding of malachite green, a cationic triphenylmethane dye, to the G-quadruplex of d[(T₂AG₃)₄] by UV spectroscopy of thermal melting analysis, a competitive equilibrium dialysis assay, and absorption and circular dichroism spectroscopies. When binding to malachite green, the quadruplex structure that formed in the presence of K⁺ ions was stabilised with an increase in melting temperatures by 6 °C. Malachite green showed selective binding to the G-quadruplex in the presence of duplex and single-stranded DNAs, owing to which it presents higher potential for anticancer therapy, compared to other triphenylmethane dyes. The induced signals of circular dichroism indicate that the binding mode of malachite green involves intercalation between adjacent guanine tetrads of the G-quadruplex.     

新型水溶性不对称五甲川菁染料的合成、光稳定性及蛋白质的荧光标记

合成并表征了系列水溶性五甲川菁染料, 研究了其在不同溶剂中的光谱性能. 结果表明, 染料在水中的最大吸收和荧光光谱在647~665 nm波长范围内, 荧光量子产率达到0.1左右. 考察了N位取代基对染料水溶液光稳定性的影响, 结果表明, 在N原子上引入带有苯环结构和大体积的磺酸基, 可以提高染料的光稳定性. 高效液相色谱(HPLC)分析结果表明, 染料4a的N-羟基琥珀酰亚胺(NHS)活性酯标记牛血清白蛋白(BSA)的检测限为1.2×10^-8 mol/L, 与紫外检测相比, 检测灵敏度提高了近2个数量级.     

Cyanine dyes as protectors of K562 cells from photosensitized cell damage

Several cyanine dyes were found to protect K562 leukemia cells against toxicity mediated by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS2) and light. Most cyanine dyes derived from dimethylindole were better photoprotectors than cyanine dyes with other structures. This correlated with the fact that cyanine dyes derived from dimethylindole were predominately monomeric at millimolar concentrations within K562 cells, while other cyanine dyes formed aggregates. For cyanine dyes that are derived from dimethylindole and have absorption band wavelengths greater than 700 nm, fluorescence-energy transfer from TPPS2 to the cyanine dye was the most important mechanism for photoprotection. There was no spectroscopic evidence for complex formation between the cyanine dyes and TPPS2. The dimethylindole derivative, 1,1',3,3,3',3'-hexamethylindodicarbocyanine, was an excellent photoprotector, but a poor quencher of TPPS2 fluorescence and a relatively poor singlet-oxygen quencher. This cyanine dye may act by quenching excited triplet TPPS2. Singlet-oxygen quenching may contribute to the photoprotection provided by cyanine dyes not derived from dimethylindole. Differences in the subcellular distribution of the various cyanine dyes studied may have contributed to the different apparent mechanisms of photoprotection.     

Duplex dissociation of telomere DNAs induced by molecular crowding

Because of the importance of telomere DNAs, the structures of these DNAs in vivo are currently of great research interest in the medical, pharmaceutical, chemical, and industrial fields. To understand the structure of biomolecules in vivo, their properties studied in vitro are extrapolated to the in vivo condition, while the condition in a living cell is inherently molecularly crowded and a nonideal solution contains various biomolecules. We investigated the effect of molecular crowding, which is one of the most important cellular environmental conditions, on the structure and stability of the telomere and G-rich and C-rich DNAs using circular dichroism (CD) spectra, CD melting curves, and isothermal titration calorimetry (ITC). The CD spectra and CD melting curves of G-rich DNA, C-rich DNA, and the 1:1 mixture of G-rich and C-rich DNAs showed that each G-rich DNA, C-rich DNA, and the 1:1 mixture form the antiparallel G-quadruplex, I-motif, and duplex, respectively, in the noncrowding condition as previously considered. On the contrary, the G-rich and C-rich DNAs individually form the parallel G-quadruplex and I-motif, respectively, in the molecular crowding condition, and the 1:1 mixture folds into the parallel G-quadruplex and I-motif but does not form a duplex. The ITC measurements indicated that the thermodynamic stability (DeltaG degrees (20)) of the duplex formation between the G-rich and C-rich DNAs in the noncrowding condition was -10.2 kcal mol(-)(1), while only a small heat change was observed in the ITC measurements in the molecular crowding condition. These ITC results also demonstrated that the molecular crowding condition prevents any duplex formation between G-rich and C-rich DNAs. These results indicate that a structural polymorphism of the telomere DNAs is induced by molecular crowding in vivo.     

Doubly Strapped Zwitterionic NIR-I and NIR-II Heptamethine Cyanine Dyes for Bioconjugation and Fluorescence Imaging

Heptamethine cyanine dyes enable deep tissue fluorescence imaging in the near infrared (NIR) window. Small molecule conjugates of the benchmark dye ZW800-1 have been tested in humans. However, long-term imaging protocols using ZW800-1 conjugates are limited by their instability, primarily because the chemically labile C4′-O-aryl linker is susceptible to cleavage by biological nucleophiles. Here, we report a modular synthetic method that produces novel doubly strapped zwitterionic heptamethine cyanine dyes, including a structural analogue of ZW800-1 , with greatly enhanced dye stability. NIR-I and NIR-II versions of these doubly strapped dyes can be conjugated to proteins, including monoclonal antibodies, without causing undesired fluorophore degradation or dye stacking on the protein surface. The fluorescent antibody conjugates show excellent tumor-targeting specificity in a xenograft mouse tumor model. The enhanced stability provided by doubly strapped molecular design will enable new classes of in vivo NIR fluorescence imaging experiments with possible translation to humans.     

Chiral transformation of cyanine dye aggregates induced by small peptides

Three small peptides (K4, K5, and K6) with different length were designed to induce the transformation of the assembled state and the chirality of cyanine dye supramolecule. The absorption and circular dichroism (CD) results indicated that, the peptides tend to induce cyanine dye to H-aggregation, competed with Na(+) in PBS, which would induce dye to J-aggregation. Meanwhile, all three peptides could influence the chirality of both J-aggregates induced by Na(+) and H-aggregates, among which K6 could induce chiral reversion of J-aggregates. Furthermore, molecular modeling and energy calculation results have shown that the peptides with different chain length have different conformations. This might be the reason for cyanine dye to form the different chiral assembly induced by these oligo-peptide templates.     

Coherent interactions of dyes assembled on DNA

The optical behavior of an organized dye assembly is different from that of the isolated dye; this difference is explained using molecular exciton theory. The theory predicts that mutual orientation, the number of dyes in the cluster, and combinations of different dyes should display given characteristic spectroscopic behaviors due to coherent interactions. Comparison of theoretical predictions with experimental results has been limited so far. One of the reasons is the absence of a rigid and well-organized system that can control the orientation and size of the dye assembly. Recently, the DNA duplex has been used to assemble chromophores in a programmed manner. Use of DNA allows organized dye assembly with a given size and particular orientation. In this review, we evaluate the spectroscopic behavior of the H-type aggregate based on molecular exciton theory and compare it with actual dye assembly with DNA duplex. Furthermore, we demonstrate the importance of coherent interactions on the observed optical properties of dyes assembled in a DNA duplex.     

Photobleaching of asymmetric cyanines used for fluorescence imaging of single DNA molecules

The photobleaching of the cyanine dyes YO and YOYO has been investigated for both free and DNA-bound dyes, using absorption and fluorescence spectroscopy coupled with fluorescence microscopy. For the free dyes, the nature of the reactive species involved in the photodegradation process is different for the monomer and the dimer, as shown by scavenger studies. For DNA-bound dyes, photoinduced fading of the visible absorption band occurs by different pathways depending on the drug binding mode and can be attenuated by appropriate scavengers. However, none of these scavengers were found to have any significant effect on the photobleaching of dye fluorescence. It appears that the reduction of fluorescence intensity comes from a quenching of the dye fluorescence by modified DNA bases, possibly 8-oxo-7,8-dihydro-2'-deoxyguanosine.     

New asymmetric monomethine cyanine dyes for nucleic-acid labelling: absorption and fluorescence spectral characteristics

Absorption spectra and fluorescence properties of a series of newly synthesized asymmetric monomethine cyanine dyes are studied. The dyes carry one or two positive charges. They are devoid of their own fluorescence in solution and become fluorescent upon binding to nucleic acids only. The fluorescence maxima of the new dyes are localized between 530 and 650 nm. The wavelength and intensity of fluorescence are dependent on molecular structure of the dye, type of nucleic acid and the concentration of both nucleic acid and salts. Some of the dyes are capable of distinguishing between single-stranded and double-stranded (ds) polynucleotides giving fluorescence maxima localized at different wavelengths. Detection threshold for dsDNA for most of the dyes is comparable to that of ethidium bromide. The sensitivity of the dye-dsDNA complexes to NaCl concentrations show that the new dyes interact with dsDNA by both intercalation and electrostatically.     

Triphenylmethane dyes as fluorescent probes for G-quadruplex recognition

Triphenylmethane (TPM) dyes normally render rather weak fluorescence due to easy vibrational deexcitation. However, when they stack onto the two external G-quartets of a G-quadruplex (especially intramolecular G-quadruplex), such vibrations will be restricted, resulting in greatly enhanced fluorescence intensities. Thus, TPM dyes may be developed as sensitive G-quadruplex fluorescent probes. Here, fluorescence spectra and energy transfer spectra of five TPM dyes in the presence of G-quadruplexes, single- or double-stranded DNAs were compared. The results show that the fluorescence spectra of four TPM dyes can be used to discriminate intramolecular G-quadruplexes from intermolecular G-quadruplexes, single- and double-stranded DNAs. The energy transfer fluorescence spectra and energy transfer fluorescence titration can be used to distinguish G-quadruplexes (including intramolecular and intermolecular G-quadruplexes) from single- and double-stranded DNAs. Positive charges and substituent size in TPM dyes may be two important factors in influencing the binding stability of the dyes and G-quadruplexes.     

Gaining Insights on the Interactions of a Class of Decorated (2-([2,2′-Bipyridin]-6-yl)phenyl)platinum Compounds with c-Myc Oncogene Promoter G-Quadruplex and Other DNA Structures

Organometallic molecules offer some of the most promising scaffolds for interaction with G-quadruplex nucleic acids. We report the efficient synthesis of a family of organoplatinum(II) complexes, featuring a 2-([2,2′-bipyridin]-6-yl)phenyl tridentate (N^∧N^∧C) ligand, that incorporates peripheral side-chains aiming at enhancing and diversifying its interaction capabilities. These include a di-isopropyl carbamoyl amide, a morpholine ethylenamide, two enantiomeric proline imides and an oxazole. The binding affinities of the Pt-complexes were evaluated via UV-vis and fluorescence titrations, against 5 topologically-distinct DNA structures, including c-myc G-quadruplex, two telomeric (22AG) G-quadruplexes, a duplex (ds26) and a single-stranded (polyT) DNA. All compounds exhibited binding selectivity in favour of c-myc, with association constants (K_a) in the range of 2–5×10⁵ M⁻¹, lower affinity for both folds of 22AG and for ds26 and negligible affinity for polyT. Remarkable emission enhancements (up to 200-fold) upon addition of excess DNA were demonstrated by a subset of the compounds with c-myc, providing a basis for optical selectivity, since optical response to all other tested DNAs was low. A c-myc DNA-melting experiment showed significant stabilizing abilities for all compounds, with the most potent binder, the morpholine-Pt-complex, exhibiting a ΔT_m>30 °C, at 1 : 5 DNA-to-ligand molar ratio. The same study implied contributions of the diverse side-chains to helix stabilization. To gain direct evidence of the nature of the interactions, mixtures of c-myc with the four most promising compounds were studied via UV Resonance Raman (UVRR) spectroscopy, which revealed end-stacking binding mode, combined with interactions of side-chains with loop nucleobase residues. Docking simulations were conducted to provide insights into the binding modes for the same four Pt-compounds, suggesting that the binding preference for two alternative orientations of the c-myc G-quadruplex thymine ‘cap’ (‘open’ vs. ‘closed’), as well as the relative contributions to affinity from end-stacking and H-bonding, are highly dependent on the nature of the interacting Pt-complex side-chain.     

Iridium (I) and Platinum (II) Complexes with Cyanine Dye Base Ligands

The preparation of cyanine dyes by coordination of cyanine dye bases with transition metals is described. The significance of these complexús for the realization of an excitonic high temperature superconductor is discussed.     

Self-Assembled Peptidyl Aggregates for the Fluorogenic Recognition of Mitochondrial DNA G-Quadruplexes

The selective monitoring of G-quadruplex (G4) structures in living cells is important to elucidate their functions and reveal their value as diagnostic or therapeutic targets. Here we report a fluorogenic probe ( CV2 ) able to selectively light-up parallel G4 DNA over antiparallel topologies. CV2 was constructed by conjugating the excimer-forming CV dye with a peptide sequence (l -Arg-l -Gly-glutaric acid) that specifically recognizes G4s. CV2 forms self-assembled, red excimer-emitting nanoaggregates in aqueous media, but specific binding to G4s triggers its disassembly into rigidified monomeric dyes, leading to a dramatic fluorescence enhancement. Moreover, selective permeation of CV2 stains G4s in mitochondria over the nucleus. CV2 was employed for tracking the folding and unfolding of G4s in living cells, and for monitoring mitochondrial DNA (mtDNA) damage. These properties make CV2 appealing to investigate the possible roles of mtDNA G4s in diseases that involve mitochondrial dysfunction.     

母核结构对三碳菁染料光氧化过程的影响

本文研究三种不同母核结构的三碳菁染料在乙醇溶剂中的光氧化反应,实验中采用了近红外区光谱能量分布平稳的光源,得出了母核结构对染料的光氧化稳定的影响,其影响次序:叮恶唑>硒唑>噻唑。同时本文还利用β值的测定,算出染料光氧化过程中敏化产生的单重态氧的浓度以及二者反应的速率常数。     

利用花青染料转化DNA杂交信号的研究

基于花青染料3-乙基-2-[7-(3-乙基-2-苯并噻唑啉)-1,3,5-庚三烯]碘化苯并噻唑与DNA杂交双链的特异性结合作用,将DNA的杂交信号转化为染料在可见光区的光吸收信号。实验结果表明,与DNA杂交双链结合后,该染料在760nm处产生强吸收峰,吸光度数值及其稳定性受杂交链长度和体系温度的影响。在25℃条件下,采用24-mer探针,可以检测mg·mL-1数量级的互补DNA链,比DNA自身杂交信号灵敏度提高两个数量级。在优化实验条件下,利用该染料还可识别短链DNA中存在的单核苷酸多态性。与其它DNA片段的检测以及单核苷酸多态性的识别方法相比,该方法更为简便迅速,在临床诊疗中具有潜在的应用价值。     

The synthesis and characterization of novel coumarin-containing cyanine dyes via “Click” chemistry

Four novel cyanine dye derivatives (C-Cy(1–4)) in which a coumarin moiety is attached to benzoindole ring of asymmetric trimethine cyanine dyes or hemicyanine dye by 1,2,3-triazole have been successfully synthesized via “Click” reaction between the corresponding alkyl-end cyanine dyes and 3-azidocoumarin. Six reaction systems were employed to find the suitable “Click” conditions of cyanine dyes. It was found that the combination of DMF, CuI and di-i-propylethylamine (DIPEA) was the most excellent system for “Click” reaction. All of the compounds were characterized by ¹H-NMR, MS, UV and FL.     

Blue fluorescent dye-protein complexes based on fluorogenic cyanine dyes and single chain antibody fragments

Fluoromodules are complexes formed upon the noncovalent binding of a fluorogenic dye to its cognate biomolecular partner, which significantly enhances the fluorescence quantum yield of the dye. Previously, several single-chain, variable fragment (scFv) antibodies were selected from a yeast cell surface-displayed library that activated fluorescence from a family of unsymmetrical cyanine dyes covering much of the visible and near-IR spectrum. The current work expands our repertoire of genetically encodable scFv-dye pairs by selecting and characterizing a group of scFvs that activate fluorogenic violet-absorbing, blue-fluorescing cyanine dyes, based on oxazole and thiazole heterocycles. The dye binds to both yeast cell surface-displayed and soluble scFvs with low nanomolar K(d) values. These dye-protein fluoromodules exhibit high quantum yields, approaching unity for the brightest system. The promiscuity of these scFvs with other fluorogenic cyanine dyes was also examined. Fluorescence microscopy demonstrates that the yeast cell surface-displayed scFvs can be used for multicolor imaging. The prevalence of 405 nm lasers on confocal imaging and flow cytometry systems make these new reagents potentially valuable for cell biological studies.     

Ionically Self-Assembled, Multi-Luminophore One-Dimensional Micro- and Nanoscale Aggregates of Thiacarbocyanine GUMBOS

Groups of uniform materials based on organic salts (GUMBOS), derived from thiacarbocyanine (TC)-based dyes with increasing methyne chain lengths, were prepared through a single-step metathesis reaction between the iodide form of the TC dye and lithium bis(perfluoroethylsulfonyl)imide as the lipophilic anion source. Ionic self-assembly of these fluorescent hydrophobic GUMBOS resulted in aqueous dispersions of one-dimensional micro-and nano-scale molecular aggregates. Blended binary and ternary aggregates containing multiple TC GUMBOS were also prepared. These nanostructures exhibited a variety of aspect ratios, affording tunable F?rster resonance energy transfer (FRET) and aggregation-dependent spectroscopic properties.     

A rainbow of fluoromodules: a promiscuous scFv protein binds to and activates a diverse set of fluorogenic cyanine dyes

Combined magnetic and fluorescence cell sorting were used to select Fluorogen Activating Proteins (FAPs) from a yeast surface-displayed library for binding to the fluorogenic cyanine dye Dimethyl Indole Red (DIR). Several FAPs were selected that bind to the dye with low nanomolar Kd values and enhance fluorescence more than 100-fold. One of these FAPs also exhibits considerable promiscuity, binding with high affinity to several other fluorogenic cyanine dyes with emission wavelengths covering most of the visible and near-IR regions of the spectrum. This significantly expands the number and wavelength range of scFv-based fluoromodules.     

Selective G-quadruplex DNA stabilizing agents based on bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine

Various biologically relevant G-quadruplex DNA structures offer a platform for therapeutic intervention for altering the gene expression or by halting the function of proteins associated with telomeres. One of the prominent strategies to explore the therapeutic potential of quadruplex DNA structures is by stabilizing them with small molecule ligands. Here we report the synthesis of bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine and their interaction with human telomeric DNA and promoter G-quadruplex forming DNAs. The interactions of ligands with quadruplex forming DNAs were studied by various biophysical, biochemical, and computational methods. Results indicated that bisquinolinium ligands bind tightly and selectively to quadruplex DNAs at low ligand concentration (～0.2-0.4 μM). Furthermore, thermal melting studies revealed that ligands imparted higher stabilization for quadruplex DNA (an increase in the T(m) of up to 21 °C for human telomeric G-quadruplex DNA and >25 °C for promoter G-quadruplex DNAs) than duplex DNA (ΔT(m) ≤ 1.6 °C). Molecular dynamics simulations revealed that the end-stacking binding mode was favored for ligands with low binding free energy. Taken together, the results indicate that the naphthyridine-based ligands with quinolinium and pyridinium side chains form a promising class of quadruplex DNA stabilizing agents having high selectivity for quadruplex DNA structures over duplex DNA structures.