Light-driven conformational regulation of human telomeric G-quadruplex DNA in physiological conditions

Human telomeric G-quadruplexes have raised broad interest not just due to their involvement in the regulation of gene expressions and telomerase activities but also because of their application in nanoarchitectures. Herein, three azobenzene derivatives 1-3 were synthesized with different substituent groups and their photo-isomerization properties were investigated by UV/Vis spectroscopy. Then circular dichroism spectroscopy (CD), fluorescence experiments and native-gel electrophoresis were performed to evaluate their capabilities of conformational photo-regulation both in the absence and presence of metal ions. The results suggested that the compounds synthesized can successfully regulate the conformation of human telomeric G-quadruplex DNA in K(+) conditions to some extent. This work will initiate the possibility for the design and intriguing application of light-induced switching to photoregulate the conformation of G-quadruplex DNA under physiological conditions, providing a possible pathway to control G-quadruplex conformation in biological applications and also expanding the potential use of G-quadruplexes in nanomachines.     

Single molecule conformational analysis of the biologically relevant DNA G-quadruplex in the promoter of the proto-oncogene c-MYC

Single molecule fluorescence spectroscopy has been employed to resolve the conformational heterogeneity, hybridization kinetics and study mutational effects on the c-MYC promoter G-quadruplex.     

A single-molecule view of conformational switching of DNA tethered to a gold electrode

Surfaces that can actively regulate binding affinities or catalytic properties in response to external stimuli are a powerful means to probe and control the dynamic interactions between the cell and its microenvironment. Active surfaces also enable novel functionalities in biosensors and biomolecular separation technologies. Although electrical stimuli are often appealing due to their speed and localization, the operation of these electrically activated surfaces has mostly been characterized with techniques averaging over many molecules. Without a molecular-scale understanding of how biomolecules respond to electric fields, achieving the ultimate detection sensitivity or localized biological perturbation with the ultimate resolution would be difficult. Using electrochemical atomic force microscopy, we are able to follow the conformational changes of individual, short DNA molecules tethered to a gold electrode in response to an applied potential. Our study reveals conformations and dynamics that are difficult to infer from ensemble measurements: defects in the self-assembled monolayer (SAM) significantly perturb conformations and adsorption/desorption kinetics of surface-tethered DNA; on the other hand, the SAM may be actively molded by the DNA at different potentials. These results underscore the importance of characterizing the systems at the relevant length scale in the development of electrically switchable biofunctional surfaces.     

Elongated thrombin binding aptamer: a G-quadruplex cation-sensitive conformational switch

Aptamer-based biosensors offer promising perspectives for high performance, specific detection of proteins. The thrombin binding aptamer (TBA) is a G-quadruplex-forming DNA sequence, which is frequently elongated at one end to increase its analytical performances in a biosensor configuration. Herein, we investigate how the elongation of TBA at its 5'?end affects its structure and stability. Circular dichroism spectroscopy shows that TBA folds in an antiparallel G-quadruplex conformation with all studied cations (Ba(2+), Ca(2+), K(+), Mg(2+), Na(+), NH(4)(+), Sr(2+) and the [Ru(NH(3))(6)](2+/3+) redox marker) whereas other structures are adopted by the elongated aptamers in the presence of some of these cations. The stability of each structure is evaluated on the basis of UV spectroscopy melting curves. Thermal difference spectra confirm the quadruplex character of all conformations. The elongated sequences can adopt a parallel or an antiparallel structure, depending on the nature of the cation; this can potentially confer an ion-sensitive switch behavior. This switch property is demonstrated with the frequently employed redox complex [Ru(NH(3))(6)](3+), which induces the parallel conformation at very low concentrations (10 equiv per strand). The addition of large amounts of K(+) reverts the conformation to the antiparallel form, and opens interesting perspectives for electrochemical biosensing or redox-active responsive devices.     

New insights from molecular dynamic simulation studies of the multiple binding modes of a ligand with G-quadruplex DNA

G-quadruplexes are higher-order DNA and RNA structures formed from guanine-rich sequences. These structures have recently emerged as a new class of potential molecular targets for anticancer drugs. An understanding of the three-dimensional interactions between small molecular ligands and their G-quadruplex targets in solution is crucial for rational drug design and the effective optimization of G-quadruplex ligands. Thus far, rational ligand design has been focused mainly on the G-quartet platform. It should be noted that small molecules can also bind to loop nucleotides, as observed in crystallography studies. Hence, it would be interesting to elucidate the mechanism underlying how ligands in distinct binding modes influence the flexibility of G-quadruplex. In the present study, based on a crystal structure analysis, the models of a tetra-substituted naphthalene diimide ligand bound to a telomeric G-quadruplex with different modes were built and simulated with a molecular dynamics simulation method. Based on a series of computational analyses, the structures, dynamics, and interactions of ligand-quadruplex complexes were studied. Our results suggest that the binding of the ligand to the loop is viable in aqueous solutions but dependent on the particular arrangement of the loop. The binding of the ligand to the loop enhances the flexibility of the G-quadruplex, while the binding of the ligand simultaneously to both the quartet and the loop diminishes its flexibility. These results add to our understanding of the effect of a ligand with different binding modes on G-quadruplex flexibility. Such an understanding will aid in the rational design of more selective and effective G-quadruplex binding ligands.     

Triplex-directed covalent cross-linking of a DNA nanostructure

The triplex approach to DNA recognition is exploited to direct covalent inter-strand cross-links to unique locations within a pre-assembled DNA nanostructure. This approach can be used to improve the stability of DNA nanostructures and demonstrates the feasibility of directing other reactive groups to unique locations within these complexes.     

pH-dependent conformational switching in 2,6-benzamidodiphenylacetylenes

The conformational equilibrium of a pH-dependent switch based on an intramolecularly H-bonded diphenylacetylene can be predictably biased by using electron-donating or -withdrawing groups. Furthermore, protonation of the electron-donating dimethylamino group converts it into an electron-withdrawing dimethylammonium cation with a concomitant switch in conformation.     

Pb2+ induced DNA conformational switch from hairpin to G-quadruplex: electrochemical detection of Pb2+

Conformational switch from hairpin DNA to G-quadruplex induced by Pb(2+) is studied by electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)(6)](3-/4-) as the redox probe. In the presence of Pb(2+), the G-rich hairpin DNA opens the stem-loop and forms G-quadruplex structure, which gives rise to a sharp increase in the charge-transfer resistance (R(CT)) of the film reflected by the EIS. This structural change is also confirmed by circular dichroism (CD) measurements and UV-Vis spectroscopic analysis and calculated by density functional theory (DFT). On the basis of this, we develop a label-free electrochemical DNA biosensor for Pb(2+) detection. With increasing concentrations of Pb(2+), the differences in the charge-transfer resistance R(CT) before and after the Pb(2+) incubation is linearly dependent on the logarithm of Pb(2+) concentration within a range from 50 μM to 0.5 nM. The biosensor also exhibits good selectivity for Pb(2+) over other metal ions. This is a simple and label-free electrochemical method for Pb(2+) detection making use of the G-quadruplex.     

DNA G-四链体识别探针研究进展

G-四链体是一种由富含鸟嘌呤核酸序列形成的独特的二级结构,广泛分布于真核生物基因组,如端粒DNA、r DNA和一系列基因中的启动子区域。G-四链体结构对很多重要的生理过程如基因的转录、复制、重组以及保持染色体的稳定性方面具有重要作用。G-四链体的特异、高灵敏检测将为进一步了解G-四链体结构在人类细胞基因组中的分布、功能和机制奠定基础,也可能为靶向G-四链体的肿瘤治疗方法提供新的思路。因而过去几十年人们一直致力于开发设计具有高选择性和高灵敏度的G-四链体识别探针,这些探针已经广泛应用于溶液中G-四链体的识别,而且具有良好的选择性。目前也有少数探针能够直接用于检测活体G-四链体结构。本文综述了一些常见的靶向G-四链体的小分子配体,以及它们在染色体和活体细胞G-四链体检测中的应用。笔者希冀本文能为设计识别G-四链体的高性能探针,进一步实现活细胞内G-四链体的检测提供借鉴。     

Conformational conversion of DNA G-quadruplex induced by a cationic porphyrin

The interactions between cationic meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the G-quadruplex (G4) of human telomeric single-strand oligonucleotide d(TTAGGG)₂ (S12) have been investigated by means of circular dichroism (CD), UV–visible absorption and fluorescence spectroscopies. It is found that TMPyP4 can preferentially induce the conformational conversion of the G4 structure from the parallel type to the parallel/antiparallel mixture in the presence of K⁺, and that it can directly induce the formation of antiparallel G4 structure from the single-strand oligonucleotide S12 in the absence of K⁺. Furthermore, the comparable experiments of TMPyP4 with two single-strand oligonucleotides S6 d(TTAGGG) and S24 d(TAGGG(TTAGGG)₃T) in the absence of K⁺ show that TMPyP4 can also induce the formation of antiparallel G4 from S24 but not from S6, indicating that the end-loops of the G4 structure are the key factors for the formation of G4 induced by TMPyP4.     

Development of a light-up fluorescent probe for HRAS G-quadruplex DNA

The development of small-molecule G-quadruplex DNA probes has attracted significant attention in recent years. However, G-quadruplexes can display a wide variety of topologies, which process different structures and functions. Therefore, selective discrimination one G-quadruplex structure over another is promising. Herein, we reported the design, synthesis and biological evaluation of a long-chain fatty amine functionalized triphenylamine-quinolinium conjugate 1b. Significant enhancement of the fluorescence intensity (over 180 fold) was observed when 1b bound with HRAS G-quadruplex DNA, while much weaker enhancements were presented in the presence of other G-quadruplexes (45–90-fold) and single/double-stranded DNAs (less than 20-fold), indicating 1b had an excellent selectivity to HRAS. The details of the interactions were investigated by UV–Vis, FID and CD analysis. The results show 1b could interact and stabilize HRAS structure mainly by π-π stacking binding mode. The introduced amine chain of the structure core was found to be better in the terms of inducing selectivity toward G-quadruplex structure. In addition, the application of 1b as a fluorescent agent for living cell imaging was also demonstrated.     

Atomic force microscopy of DNA at high humidity: irreversible conformational switching of supercoiled molecules

Three topologically different double-stranded DNA molecules of the same size (bps) have been imaged in air on mica using amplitude modulation atomic force microscopy (AM AFM) under controlled humidity conditions. At very high relative humidity (>90% RH), localized conformational changes of the DNA were observed, while at lower RH, the molecules remained immobile. The conformational changes occurred irreversibly and were driven principally by superhelical stress stored in the DNA molecules prior to binding to the mica surface. The binding mechanism of the DNA to the mica (surface equilibration versus kinetic trapping) modulated the extent of the conformational changes. In cases where DNA movement was observed, increased kinking of the DNA was seen at high humidity when more surface water was present. Additionally, DNA condensation behavior was also present in localized regions of the molecules. This study illustrates that changes in the tertiary structure of DNA can be induced during AFM imaging at high humidity on mica. We propose that AM AFM in high humidity will be a useful technique for probing DNA topology without some of the drawbacks of imaging under bulk solution.     

邻菲罗啉衍生物与G-四链体DNA的识别

设计合成了一种新型邻菲罗啉衍生物,用IR,<'1>H-NMR,<'13>C-NMR和质谱进行了结构表征.在包含100mM NaCl的Tris-HCl(pH 7.4)缓冲溶液中,利用荧光共振能量转移熔点(FRET-melting)分析以及紫外可见光谱研究了此化合物对人端粒G-四链体DNA(AG<,3>T<,2>AG<,3...