Methods for investigating G-quadruplex DNA/ligand interactions

DNA is considered an important target for drug design and development. Until recently, the focus was on double-stranded (duplex) DNA structures. However, it has now been shown that single stranded DNA can fold into hairpin, triplex, i-motif and G-quadruplex structures. The more interesting G-quadruplex DNA structures comprise four strands of stacked guanine (G)-tetrads formed by the coplanar arrangement of four guanines, held together by Hoogsteen bonds. The DNA sequences with potential to form G-quadruplex structures are found at the chromosomal extremities (i.e. the telomeres) and also at the intra-chromosomal region (i.e. oncogenic promoters) in several important oncogenes. The formation of G-quadruplex structures is considered to have important consequences at the cellular level and such structures have been evoked in the control of expression of certain genes involved in carcinogenesis (c-myc, c-kit, K-ras etc.) as well as in the perturbation of telomeric organization. It has been shown that the formation of quadruplexes inhibits the telomere extension by the telomerase enzyme, which is up-regulated in cancer cells. Therefore, G-quadruplex structures are an important target for drug design and development and there is a huge interest in design and development of small molecules (ligands) to target these structures. A large number of so-called G-quadruplex ligands, displaying varying degrees of affinity and more importantly selectivity (i.e. the ability to interact only with quadruplex-DNA and not duplex-DNA), have been reported. Access to efficient and robust in vitro assays is needed to effectively monitor and quantify the G-quadruplex DNA/ligand interactions. This tutorial review provides an overview of G-quadruplex ligands and biophysical techniques available to monitor such interactions.     

A pyrazine-based fluorescence-enhancing ligand with a high selectivity for thymine in AP site-containing DNA duplexes

A fluorescent pyrazine derivative, 3,5-diamino-6-chloro-2-pyrazine carbonitrile (DCPC), is presented as a promising light-up ligand for single-nucleotide polymorphisms (SNPs) typing. In solutions buffered to pH 7.0 (I = 0.11 M, at 5 degrees C), DCPC can bind to thymine selectively over other nucleobases opposite an abasic site in DNA duplexes (5'-GTGTG CGTTG ANA TGGAC GCAGA-3'/3'-CACAC GCAAC TXT ACCTG CGTCT-5', X = abasic site, N = target nucleotide) with a dissociation constant of 2.6 microM. The binding of DCPC is accompanied by a significant enhancement of its fluorescence (lambda(max), 412 nm), and the response is highly selective to thymine base. These binding and sensing properties allow a clear detection of thymine-related mutations present in polymerase chain reaction (PCR) amplification products.     

A lead-filled G-quadruplex: insight into the G-Quartet's selectivity for Pb(2+) over K(+)

The lipophilic nucleoside, G 1, extracts Pb(2+) picrate from water into organic solvents to give structures based on the hydrogen-bonded G-quartet. Crystal structures indicate important differences between (G 1)(8)-Pb(2+) and (G 1)(8)-K(+). The divalent Pb(2+) templates a smaller G(8) cage than does K(+), as judged by the M-O6 bond length, O6-O6 diagonal distance, and inter-tetramer separation. The more compact Pb(2+) octamer correlates with NMR data indicating that N2-N7 hydrogen bonds in (G 1)(8)-Pb(2+) are kinetically more stable than in (G 1)(8)-K(+).     

The role of thermodynamics and kinetics in ligand binding to G-quadruplex DNA

Molecular dynamics simulations were used to investigate the binding of four different 2,4,6-triarylpyridines to G-quadruplex DNA. Both the binding free energies, and the kinetics of binding are required to explain the measured degree of ligand induced stabilisation of the compounds, with bulky substituents having the potential to prevent the ligand from reaching the lowest energy binding site.     

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.     

Synthesis of distamycin A polyamides targeting G-quadruplex DNA

A number of amide-linked oligopyrroles based on distamycin molecules have been synthesized by solid-state methods, and their interactions with a human intramolecular G-quadruplex have been measured by a melting procedure. Several of these molecules show an enhanced ratio of quadruplex vs. duplex DNA binding compared to distamycin itself, including one with a 2,5-disubstituted pyrrole group. Quadruplex affinity increases with the number of pyrrole groups, and it is suggested that this is consistent with a mixed groove/G-quartet stacking binding mode.     

A DNA G-quadruplex converts SOD1 into fibrillar aggregates

Nucleic acids with G4 elements play a role in the formation of aggregates involved in intracellular phase transitions. Our previous studies suggest that different forms of DNA could act as an accelerating template in Cu/Zn superoxide dismutase (SOD1) aggregation. Here, we examined the regulation of formation and cytotoxicity of the SOD1 aggregates by single-stranded 12-mer deoxynucleotide oligomers (dN)₁₂ (N = A, T, G, C; ssDNAs) under acidic conditions. The ssDNAs can be divided into two groups based on their roles in SOD1 binding, exposure of hydrophobic clusters in SOD1, accelerated formation, morphology and cytotoxicity of SOD1 aggregates. G-quadruplexes convert SOD1 into fibrillar aggregates as a template, a fact which was observed for the first time in the nucleic acid regulation of protein aggregation. Moreover, the fibrillar or fibril-like SOD1 species with a G-quadruplex provided by (dG)₁₂ were less toxic than the amorphous species with (dN)₁₂ (N = A, T). This study not only indicates that both morphology and cytotoxicity of protein aggregates can be regulated by the protein-bound DNAs, but also help us understand roles of nucleic aid G-quadruplexes in the formation of aggregates and membraneless organelles involved in intracellular phase transitions.     

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.     

G-quadruplex DNA and ligand interaction in living cells using NMR spectroscopy

Gathering structural information from biologically relevant molecules inside living cells has always been a challenging task. In this work, we have used multidimensional NMR spectroscopy to probe DNA G-quadruplexes inside living Xenopus laevis oocytes. Some of these structures can be found in key regions of chromosomes. G-quadruplexes are considered potential anticancer therapeutic targets and several lines of evidence indirectly point out roles in key biological processes, such as cell proliferation, genomic instability or replication initiation. However, direct demonstrations of the existence of G-quadruplexes in vivo are scarce. Using SOFAST-HMQC type spectra, we probed a tetramolecular G-quadruplex model made of d(TG₄T)₄ inside living Xenopus laevis oocytes. Our observations lead us to conclude that the quadruplex structure is formed within the cell and that the intracellular environment preferentially selects a conformation that most resembles the one found in vitro under KCl conditions. We also show for the first time that specific ligands targeting G-quadruplexes can be studied using high resolution NMR directly inside living cells, opening new avenues to study ligand binding discrimination under physiologically relevant conditions with atomic detail.     

A dumbbell double nicked duplex dodecamer DNA with a PEG6 tether

A hairpin dodecamer DNA motif with a dangling end composed of four bases was studied in order to find conditions which promote a dumbbell structure as the sole form in solution. It could be used as a model of a DNA duplex with two nicks on opposite strands, mimicking a target for topo II poisons. We have established two alternative means of obtaining a dumbbell in solution as the only form present at 0 °C. The first one is to use a relatively high concentration of a hairpin motif, ca. 3.5 mM, at low ionic strength, and second is to use a moderate hairpin motif concentration of ca. 2 mM at high ionic strength, 200 mM and 15% of methanol. An NMR-derived structure in a buffered water solution is presented. A representative structure ensemble of 10 structures was obtained from MD calculations utilizing the AMBER protocol and using NOESY-derived experiment cross peak volumes transferred to experimental restraints by the MARDIGRAS algorithm.     

A fluorescein-based probe with high selectivity to cysteine over homocysteine and glutathione

A fluorescent probe based on fluorescein displays excellent selectivity and sensitivity for cysteine and its application for bio-imaging is described.     

Dithiodiglycolamide: novel ligand with highest selectivity and extractability for palladium

A novel multidentate ligand N,N,N′,N′-tetra(2-ethylhexyl) dithiodiglycolamide DTDGA has been synthesized and studied for its extraction behavior towards various elements present in high level liquid waste (HLW). The extractant showed remarkable extractability and selectivity for palladium vis a vis other metal ions present in HLW. The distribution ratio as well as the separation factor for palladium was found to be the highest reported so far thus making this extractant one of the most promising candidates for effective separation of palladium from HLW.     

A computational tool to optimize ligand selectivity between two similar biomacromolecular targets

Summary Algorithms for a new computer program designed to increase ligand--receptor selectivity between two proteins are described. In this program ligand--receptor selectivity is increased by functional modifications to the ligand so as to increase the calculated binding affinity of it to one protein and/or decrease the calculated binding affinity of it to the other protein. The structure of the ligand is modified by selective replacement of atoms and/or functional groups in silico based on a specific set of steric and/or hydropathic complementarity rules involving atoms and functional groups. Relative binding scores are calculated with simple grid-based steric penalty, hydrogen bond complementarity, and with the HINT score model. Two examples are shown. First, modifying the structure of the ligand CB3717 is illustrated in a number of ways such that the binding selectivity to wild type L. casei thymidylate synthase or its E60Q mutant may be improved. Second, starting with a non-selective lead compound that had been co-crystallized with both plant and mammalian 4-hydroxyphenylpyruvate dioxygenases, new compounds (similar to selective ligands discovered by screening) to improve the selectivity of (herbicidal) inhibitors for the plant enzyme were designed by the program.     

Preparation of branched structures with long DNA duplex arms

Branched structures with long DNA duplex arms have been constructed through biotin-streptavidin binding and characterized by gel electrophoresis and atomic force microscopy (AFM) imaging.     

A structural transition in duplex DNA induced by ethylene glycol

The twist energy parameter ( E T) that governs the supercoiling free energy, and the linking difference (Delta l) are measured for p30delta DNA in solutions containing 0-40 w/v % ethylene glycol (EG). A plot of E T vs -ln a w, where a w is the water activity, displays the full (reverse) sigmoidal profile of a discrete structural transition. A general theory for the effect of added osmolyte on a cooperative structural transition between two duplex states, 1 right arrow over left arrow 2, is formulated in terms of parameters applicable to individual base-pair subunits. The resulting fraction of base pairs in the 2-state ( f 2 (0)) is incorporated into expressions for the effective torsion and bending elastic constants, the effective twist energy parameter ( E T (eff)), and the change in intrinsic twist (delta l 0). Fitting the expression for E T (eff) to the measured E T values yields reasonably unambiguous estimates of E T 1 and E T 2 , the midpoint value (ln a w) 1/2, and the midpoint slope ( partial differential E T/ partial differential ln a w) 1/2, but does not yield unambiguous estimates of the equilibrium constant ( K 0), the difference in DNA-water preferential interaction coefficient (DeltaGamma), or the inverse cooperativity parameter ( J). Fitting a noncooperative model (assumed J = 1.0) to the data yields K 0 = 0.067 and DeltaGamma = -30.0 per base pair (bp). Essentially equivalent fits are provided by models with a wide range of correlated J, DeltaGamma, and K 0 values. Other results favor DeltaGamma in the range -1.0 to 0, which then requires K 0 > or = 0.914, and a cooperativity parameter, 1/ J > or = 30.0 bp. The measured delta l 0 and circular dichroism (CD) at 272 nm are found to be compatible with curves predicted using the same f 2 (0) values that best-fit the E T data. At least 7-10% of the base pairs are inferred to exist in the 2-state in 0.1 M NaCl in the complete absence of added osmolyte. Compared with the 1-state, the 2-state has a approximately 2.0- to 2.1-fold greater torsion elastic constant, a approximately 0.70-fold smaller bending elastic constant, a approximately 0.91-fold smaller E T value, a approximately 0.2% lower intrinsic twist, a somewhat lower CD near both 272 and 245 nm, and less water and/or more EG in its neighborhood. However, the relative change in preferential interaction coefficient associated with the transition is likely rather slight.     

The prenylated dioxopiperazine alkaloid Cristatin A has selective telomeric DNA G-quadruplex stabilising properties

Cristatin A (1a/b), a prenylated dioxopiperazine alkaloid, has been shown to bind selectively to telomeric quadruplex DNA using a FRET-based DNA melting assay. Crucially, the molecule is more drug-like than most previously identified quadruplex-binding agents, and provides a unique chemical scaffold for future chemical biology and drug discovery studies.