Asymmetric Distyrylpyridinium Dyes as Red‐Emitting Fluorescent Probes for Quadruplex DNA

The interactions of three cationic distyryl dyes, namely 2,4‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 1 a ), its derivative with a quaternary aminoalkyl chain ( 1 b ), and the symmetric 2,6‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 2 a ), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA‐binding and DNA‐probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4‐disubstituted) dyes 1 a and 1 b bind to quadruplex DNA structures with a near‐micromolar affinity and a fair selectivity with respect to double‐stranded (ds) DNA [K_a(G4)/K_a(ds)=2.5–8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80–100‐fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double‐stranded DNA. This optical selectivity allows these dyes to be used as quadruplex‐DNA‐selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2 a displays a strong binding preference for double‐stranded DNA [K_a(ds)/K_a(G4)=40–100), presumably due to binding in the minor groove. In addition, 2 a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20–50‐fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA‐recognition properties and fluorimetric response of organic dyes.     

Interactions of Amino Acids with Oxidized Guanine in the Gas Phase Associated with the Protection of Damaged DNA

Density functional theory calculations were employed to study the stabilization process of the guanine radical cation through amino acid interactions as well as to understand the protection mechanisms. On the basis of our calculations, several protection mechanisms are proposed in this work subject to the type of the amino acid. Our results indicate that a series of three‐electron bonds can be formed between the amino acids and the guanine radical cation which may serve as relay stations supporting hole transport. In the three‐electron‐bonded, π–π‐stacked, and H‐bonded modes, amino acids can protect guanine from oxidation or radiation damage by sharing the hole, while amino acids with reducing properties can repair the guanine radical cation through proton‐coupled electron transfer or electron transfer. Another important finding is that positively charged amino acids (ArgH⁺, LysH⁺, and HisH⁺) can inhibit ionization of guanine through raising its ionization potential. In this situation, a negative dissociation energy for hydrogen bonds in the hole‐trapped and positively charged amino acid–Guanine dimer is observed, which explains the low hole‐trapping efficiency. We hope that this work provides valuable information on how to protect DNA from oxidation‐ or radiation‐induced damages in biological systems.     

Solution Structure of a G‐quadruplex Bound to the Bisquinolinium Compound Phen‐DC3

Phen‐DC₃ is a highly promising compound that specifically targets G‐quadruplexes, with potent biological effects observed in vivo. We used NMR spectroscopy to solve the structure of the complex formed between Phen‐DC₃ and an intramolecular G‐quadruplex derived from the c‐myc promoter. Structural information revealed that Phen‐DC₃ interacts with the quadruplex through extensive π‐stacking with guanine bases of the top G‐tetrad. On the basis of our structure, modifications are proposed for the development of this compound for selective targeting of a specific G‐quadruplex conformation.     

The influence of formamide (model of protein unit) on the intramolecular proton transfer in the DNA simple base guanine: A density functional theory study

For the purpose of investigating the influence of protein unit on the intramolecular proton transfer (IPT) reactions in the simple base guanine, a simple model (formamide) of peptides is designed to biological system investigations, and five complexes of formamide–guanine (FG1, FG2, FG3, FG4, and FG5) are determined at the B3LYP/6‐311++G(d,p) level of theory. For comparison, HF and MP2 methods are also used in this paper. The proton transfer (PT) reaction processes of guanine and FGs have been investigated employing the B3LYP/6‐311++G(d,p) level of theory. The selected thermodynamic and kinetic parameters, such as the activation energies (E_a), changes of enthalpy (ΔH) and changes of free energies (ΔG), as well as the equilibrium constants (K_p) for those reaction processes, have also been obtained by calculational means. The calculated results indicate that the assisted and protected effects of formamide on IPT in guanine are site‐dependent. CH1 is the lowest activation energy needed PT process no matter where the formamide molecule is located in. The activation energy of CH1 with formamide in S2 is the lowest one (153.3 KJ/mol), whereas the one of CH5 with formamide in S5 is the highest (318.3 KJ/mol). © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008     

Interaction of G-quadruplex Forming DNA Aptamers with PAMAM Dendrimers Studied by Dynamic Light Scattering and UV-VIS Spectrophotometry**

The complexes of G-quadruplex forming DNA thrombin binding aptamers (TBA) and polyamidoamine dendrimers (PAMAM) were studied with the aim to form a model targeted drug delivery system. Hydrodynamic diameter, zeta potential and melting temperature (T_m) were investigated by dynamic light scattering and UV-VIS spectrophotometry. Non-covalent adsorption by means of electrostatic interaction between positively charged amino groups of dendrimers (+) and negatively charged phosphate groups of aptamers (−) has driven the formation of aggregates. The size of complexes was in the range of 0.2–2 μm and depended on the type of dispersant, charge ratio (+/−) and temperature. Raising the temperature increased the polydispersity, new smaller size distributions were observed indicating the G-quadruplex unfolding. The melting transition temperature of TBA aptamer was affected by the presence of amino-terminated PAMAM rather than carboxylated succinic acid PAMAM−SAH dendrimer, thus supporting the electrostatic nature of interaction that disturbed denaturation of target-specific quadruplex aptamer structure.     

Label-free fluorescent probing of G-quadruplex formation and real-time monitoring of DNA folding by a quaternized tetraphenylethene salt with aggregation-induced emission characteristics

Biosensing processes such as molecular beacons require non-trivial effort to covalently label or mark biomolecules. We report here a label-free DNA assay system with a simple dye with aggregation-induced emission (AIE) characteristics as the fluorescent bioprobe. 1,1,2,2-Tetrakis[4-(2-bromoethoxy)phenyl]ethene is nonemissive in solution but becomes highly emissive when aggregated. This AIE effect is caused by restriction of intramolecular rotation, as verified by a large increase in the emission intensity by increasing viscosity and decreasing temperature of the aqueous buffer solution of 1,1,2,2-tetrakis[4-(2-triethylammonioethoxy)phenyl]ethene tetrabromide (TTAPE). When TTAPE is bound to a guanine-rich DNA strand (G1) via electrostatic attraction, its intramolecular rotation is restricted and its emission is turned on. When a competitive cation is added to the G1 solution, TTAPE is detached and its emission is turned off. TTAPE works as a sensitive poststaining agent for poly(acrylamide) gel electrophoresis (PAGE) visualization of G1. The dye is highly affinitive to a secondary structure of G1 called the G-quadruplex. The bathochromic shift involved in the G1 folding process allows spectral discrimination of the G-quadruplex from other DNA structures. The strong affinity of TTAPE dye to the G-quadruplex structure is associated with a geometric fit aided by the electrostatic attraction. The distinct AIE feature of TTAPE enables real-time monitoring of folding process of G1 in the absence of any pre-attached fluorogenic labels on the DNA strand. TTAPE can be used as a K+ ion biosensor because of its specificity to K+-induced and -stabilized quadruplex structure.     

DNA Hydrolysis and Voltammetric Determination of Guanine and Adenine Using Different Electrodes

Abstract

This work reports the development of a biosensor method for the label‐free detection of specific DNA sequences. In the initial phase, square wave voltammetry (SWV) was used in a comparative investigation into the electrochemical oxidation of purines (guanine and adenine) and DNA fragments at various electrode surfaces: carbon paste (CPE), glassy carbon electrode (GCE), and gold (AuE). Relative to the carbon electrodes, an approximate 4.0‐fold, 6.0‐fold, and 3.25‐fold increase in the anodic response was observed when guanine, adenine, and hydrolyzed DNA, respectively, were measured on the AuE. It was shown that the guanine and adenine bases could be successfully determined by use of SWV for a deoxyribonucleic acid sample following acid hydrolysis. This label‐free detection of hydrolyzed DNA on gold electrodes has significant advantages over methods using existing carbon electrode materials because of its higher sensitivity and the potential applicability of microfabrication techniques for the production of the requisite gold electrodes.

In another phase of development, the times and conditions for DNA hydrolysis and purine release were investigated. It was shown that under optimal conditions, trace levels of the purine bases could be readily detected following 20 min of hydrolysis at room temperature. The proposed method can be used to estimate the guanine and adenine contents in DNA with in a linear range of 5–30 ng ml^?1.

Finally, when appropriate probe sequences were first adsorbed on the surface of the screen‐printed gold electrode (SPGE), this electrochemical biosensor could be used to specifically detect sequences from ss corona virus aviair following hybridization and hydrolysis reactions on the sensor surface. No enhancement of the voltammetric response was observed when the sensor was challenged with a non‐complementary DNA sequence.     

G-rich sequence-specific recognition and scission of human genome by PNA/DNA hybrid G-quadruplex formation

Hole in one: A single peptide nucleic acid (PNA) effectively targets the G-rich region in double-stranded DNA through formation of a PNA/DNA hybrid G-quadruplex. Only one target site in the whole human genome was selectively cleaved by the hybrid G-quadruplex. Such site-selective scission of DNA is central to gene manipulation for molecular biology, biotechnology, and therapy.     

Syntheses,Characterization, and DNA Binding Studies of a Series of Copper(II), Nickel(II), Platinum(II) and Zinc(II) Complexes Derived from Schiff Base Ligands

Three series of metal salophen complexes derived from Zn²⁺, Cu²⁺, Pt²⁺ and Ni²⁺ have been synthesized and their interaction with quadruplex DNA has been evaluated. The compounds differ on the number of ethyl piperidine substituents. They have been characterized by ¹H NMR, IR and UV-visible spectroscopies and by HR-mass spectrometry. Their luminescent properties have been also evaluated and we can observe that, as expected, Zn²⁺ and Pt²⁺ complexes are those displaying more interesting luminescence with an emission band red-shifted with respect to the corresponding uncoordinated ligand. DNA interactions with G4 and duplex DNA were evaluated by FRET melting assays (for the Zn²⁺, Cu²⁺ and Ni²⁺ complexes) and by emission titrations (for one Pt²⁺ complex) which indicated that the disubstituted compounds 2-Ni and 2-Pt are the only ones that display good affinity for G4 DNA structures.