Fluorescence properties of 2-aminopurine in human telomeric DNA

The substitution of 2-aminopurine (Ap) for A7 in the human telomeric sequence d[AGGG(TTAGGG)(3)] resulted in a significant increase in the fluorescence intensity of Ap for the conformational change from duplex to quadruplex.     

Monitoring G-quadruplex structures and G-quadruplex-ligand complex using 2-aminopurine modified oligonucleotides

The fluorescent base 2-aminopurine (2Ap) was incorporated into the human telomeric DNA sequence d[AGGG(TTAGGG)₃]. The substitution of 2Ap for A in the TTA loops did not affect the G-quadruplex stability. Interestingly, a significant change in the fluorescence intensity of 2Ap between the G-quadruplex and duplex was observed. Therefore, we demonstrated that 2Ap can be used to monitor the duplex to quadruplex conformational change in the human telomeric DNA sequence. This mechanism is explained by the difference in the base stacking in the TTA loop region. Moreover, these probes distinguished between the basket-type and propeller-type G-quadruplexes. We also demonstrated the detection of the telomerase inhibitor agent, such as TMPyP4, using a 2Ap modified telomeric DNA. The formation of the G-quadruplex-ligand complex was observed by the fluorescence titration of TMPyP4.     

Stabilisation of human telomeric quadruplex DNA and inhibition of telomerase by a platinum-phenanthroline complex

Two new mono-substituted phenanthroline ligands and their platinum(II) square planar complexes have been prepared; one of the complexes has been shown to induce a high degree of quadruplex DNA stabilisation and to inhibit telomerase.     

DNA base stacking involving adenine and 2-aminopurine

The potential energy surfaces of stacked structures consisting of adenine (A) and 2-aminopurine (2AP) have been investigated in the gas phase. Both face-to-back (the double-ring system of one base exactly on top of that of the other one) and face-to-face (one base flipped by 180°) A/A, 2AP/2AP and A/2AP stacks were considered. Minima and transition states were optimised at the counterpoise-corrected M06-2X/6-31+G(d) level of theory. For each type of stack, between five and nine minima were located, usually connected by low barriers of 1–2 kcal/mol. This shows the large conformational flexibility of these stacked base pairs. The double-ring system in A and 2AP affords multiple minima with similar twist angles, making the potential energy surface of stacks comprising of purine bases more complex than those of pyrimidine stacks. The locations of the stationary points on the potential energy surface differ for the three different systems; thus, the replacement of A by 2AP in a base stack changes its potential energy landscape.     

Telomestatin-induced stabilization of the human telomeric DNA quadruplex monitored by electrospray mass spectrometry

Electrospray mass spectrometry (ESI-MS) was used to monitor the kinetics of duplex formation between the human telomeric DNA quadruplex and its complementary strand; the complexation of telomestatin to the G-quadruplex delays the unwinding of the quadruplex structure and formation of the duplex.     

Structure of human telomeric DNA in crowded solution

G-quadruplex structures formed by DNA at the human telomeres are attractive anticancer targets. Human telomeric sequences can adopt a diverse range of intramolecular G-quadruplex conformations: a parallel-stranded conformation was observed in the crystalline state, while at least four other forms were seen in K(+) solution, raising the question of which conformation is favored in crowded cellular environment. Here, we report the first NMR structure of a human telomeric G-quadruplex in crowded solution. We show that four different G-quadruplex conformations are converted to a propeller-type parallel-stranded G-quadruplex in K(+)-containing crowded solution due to water depletion. This study also reveals the formation of a new higher-order G-quadruplex structure under molecular crowding conditions. Our molecular dynamics simulations of solvent distribution provide insights at molecular level on the formation of parallel-stranded G-quadruplex in environment depleted of water. These results regarding human telomeric DNA can be extended to oncogenic promoters and other genomic G-rich sequences.     

人类端粒DNA的分子识别及其作用机制探讨

核酸中富含短的G-碱基重复的序列可以形成一种复杂的高级结构,称为G-四链体（G-quadruplex）.在基因组中,借助生物信息学发现这类富G序列广泛分布在基因的启动子区,特别是那些参与到复制中去的基因,例如癌基因.同时发现这类序列在mRNA的5′非翻译区（5′UTR）也广泛存在.这类序列在染色体末段端粒部位的存在及功能已得到充分阐明.已知端粒富含G-碱基序列,其3′末端以单链状态存在,这使得在一些小分子的选择性作用下端粒序列很容易形成G-四链体结构,进而破坏端粒结构,影响端粒酶活性.已知端粒酶在超过85%的肿瘤中过量表达,因此,端粒酶已经成为抗癌药物设计的特殊靶点,是目前本领域的研究热点之一.已发现系列配体通过有效抑制端粒酶而表现高的抗肿瘤活性.本文主要综述了近年来端粒G-四链体分子识别及其药物靶向的最新进展,并对其作用机理做了进一步的分析和探讨.     

Ultrafast site-specific fluorescence quenching of 2-aminopurine in a DNA hairpin studied by femtosecond down-conversion

The ΔP(-)PBS analog of the DNA primary binding sequence (PBS) of the HIV-1 genome labeled at different positions by 2-aminopurine (2-AP) is investigated by a novel femtosecond fluorescence down-conversion experiment with 0.3-ps time resolution. The high signal-to-noise ratio of the fluorescence kinetics makes it possible to reveal four distinct decay times ranging from 0.8 ps to 2-3 ns for all the three labeling positions. This suggests the existence of at least four different quenching conformations of 2-AP with its nearest neighbors, and underscores the structural heterogeneity of the loop region of ΔP(-)PBS. Sub-5-ps components are found and attributed to stacking interactions of 2-AP with the flanking guanine (G) side chains, consistent with the NMR structure of ΔP(-)PBS. The observation of a significant increase of their total amplitude when 2-AP is positioned close to the rigid 3'-half of the G-rich stem gives further support to this assignment. Only a minor portion of conformations involves slow nanosecond collisional quenching.     

Enantioselective Friedel-Crafts reactions in water catalyzed by a human telomeric G-quadruplex DNA metalloenzyme

A human telomeric G-quadruplex (G4DNA) metalloenzyme, assembled with G4DNA and Cu(2+) ions, can catalyze the enantioselective Friedel-Crafts (F-C) reaction in water with good enantioselectivity (up to 75% ee). Furthermore, we found that the absolute configuration and the enantioselectivity of the product largely depend on the conformation and the sequence of G4DNA.     

Mechanism of intram olecular charge transfer in DNA helix as probed by the use of the fluorescent 2-aminopurine

~~Mechanism of intram olecular charge transfer in DNA helix as probed by the use of the fluorescent 2-aminopurine~~     

Energetic basis of human telomeric DNA folding into G-quadruplex structures

Recent theoretical studies performed on the folding/unfolding mechanism of the model telomeric human DNA, 5'-AGGGTTAGGGTTAGGGTTAGGG-3' (Tel22), have indicated that in the presence of K(+) ions Tel22 folds into two hybrid G-quadruplex structures characterized by one double and two reversal TTA loops arranged in a different way. They predicted a new unfolding pathway from the initial mixture of hybrid G-quadruplexes via the corresponding intermediate triplex structures into the final, fully unfolded state. Significantly, no experimental evidence supporting the suggested pathway has been reported. In the current work, we performed a comprehensive global thermodynamic analysis of calorimetric (DSC, ITC) and spectroscopic (CD) data obtained on monitoring the folding/unfolding of Tel22 induced by changes of temperature and K(+) concentration. We show that unfolding of Tel22 may be described as a monomolecular equilibrium three-state process that involves thermodynamically distinguishable folded (F), intermediate (I), and unfolded (U) state. Considering that calorimetric methods cannot distinguish between energetically similar G-quadruplex or triplex conformations predicted by the theoretical model one can conclude that our results represent the first experimental support of the suggested unfolding/folding mechanism of Tel22. This conclusion is confirmed by the fact that the estimated number of K(+) ions released upon each unfolding step in our thermodynamic model agrees well with the corresponding values predicted by the theoretical model and that the observed changes in enthalpy, entropy, and heat capacity accompanying the F → I and I → U transitions can be reasonably explained only if the intermediate state I is considered to be a triplex structural conformation.     

Kinetics of unfolding the human telomeric DNA quadruplex using a PNA trap

The kinetics of opening of the DNA quadruplex formed by the human telomeric repeat have been investigated using real-time fluorescence resonance energy transfer (FRET) measurements with a peptide nucleic acid (PNA) trap. It has been found that this opening is zero-order with respect to PNA, indicating that the initial step is a rate-limiting internal rearrangement of the quadruplex. A study of the temperature dependence of the rate of quadruplex opening was performed and the activation energy of the process estimated to be 98 +/- 8 kJ mol(-1).     

Selective guanine oxidation by UVB-irradiation in telomeric DNA

The combination of the transient absorption measurement and DNA damage quantification by HPLC clearly demonstrated the preferential excitation of guanine and its decomposition in quadruplex DNA by UVB-irradiation.     

Multivariate investigation of interaction between porphyrin ligands and human telomeric DNA

G-quadruplexes are formed by association of DNA strands containing multiple contiguous guanines. The capability of drugs to induce formation or stabilize G-quadruplexes is an active area of cancer therapy investigation. We evaluated interaction between two cationic tetrapyridinoporphyrazines with Na⁺ and K⁺ forms of human telomeric G-quadruplex DNA by chemometrics method. An antiparallel quadruplex structure was found to be stabilized more greatly by these two isomers in the presence of K⁺ and Na⁺ ions. Equilibrium model of a ligand binding with DNA oligomer has been considered as a process of small molecule adsorption on to a lattice of multiple binding sites. In multivariate analysis methods, it is accounted this assertion that during saturation of the macromolecule by a ligand should expect effect of cooperativity due to changes in DNA conformation or the mutual influence between bound ligands. Such phenomenon cannot be entirely described by the classical stepwise complex formation model. From the results of absorption and circular dichroism measurements, the unique site for the ligand binding is suggested to be the intercalating in guanine tetrad plane quadruplex. We found a 2:1 binding stoichiometry for both ligands and Tel22.     

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.     

Intramolecular folding in three tandem guanine repeats of human telomeric DNA

Intramolecular folding in three tandem guanine repeats of human telomeric DNA has been investigated using optical-tweezers, MD simulation and circular dichroism. A mechanically and thermodynamically stable species in this sequence shows a structure consistent with a triplex conformation. A similar species has also been observed to coexist with a G-quadruplex in a DNA sequence with four tandem guanine repeats.     

The synthesis of a menthol derivative of 2-aminopurine as a fluorescent DNA lesion

An efficient synthetic route to the phosphoramidite of a menthol functionalized guanosine analog is presented. Two procedures were executed for the key introduction of the 6′-allyl menthyl moiety. Stille vinylation on 6-O-tosylguanosine followed by cross-metathesis using an excess of allyl menthyl ether proved to be less efficient than a Stille coupling on the same tosylate using an advanced menthyl-allyl stannane derivative. Incorporation of the modified nucleoside using the phosphoramidite method into a DNA 50-mer proceeded uneventfully.     

Conformations of individual quadruplex units studied in the context of extended human telomeric DNA

G-quadruplex conformations within a sequence of three quadruplex units of human telomeric DNA were studied by two-frequency pulsed electron paramagnetic resonance (EPR) spectroscopy. In contrast to some individual G-quadruplexes, within the higher-order human telomeric sequence a (3+1) hybrid structure is formed.     

Structural heterogeneity in DNA: temperature dependence of 2-aminopurine fluorescence in dinucleotides.

The fluorescent base analogue 2-aminopurine is a sensitive probe for local dynamics of DNA. Its fluorescence is quenched by interaction with the neighboring bases, but the underlying mechanisms are still under investigation. We studied 2-aminopurine fluorescence in dinucleotides with each of the natural bases. Consistently, two of the four fluorescence-decay components depend strongly on temperature. Our results indicate that these components are due to the excited-state dynamics of a single conformational state. We propose a variation of the gating model in which transient unstacking occurs in the excited state.     

Promoting the formation and stabilization of human telomeric G-quadruplex DNA, inhibition of telomerase and cytotoxicity by phenanthroline derivatives

Four new di-substituted phenanthroline-based compounds a-d have been designed and prepared, and they have been shown to induce the formation of anti-parallel structure of human telomeric G-quadruplex DNA by CD spectra. FRET assay indicates that the melting temperature increases (ΔT(m) values) of G-quadruplex in buffer (pH 7.4) containing 100 mM NaCl are 31.6, 34.6, 17.8 and 32.6 °C for the compounds (1.0 μM) a, b, c and d, respectively. Competitive FRET assay shows that the four compounds exhibit a high G-quadruplex DNA selectivity over duplex DNA. Three of the compounds are the potent telomerase inhibitors and HeLa cell proliferation inhibitors.