Discrimination of G‐Quadruplexes from Duplex and Single‐Stranded DNAs with Fluorescence and Energy‐Transfer Fluorescence Spectra of Crystal Violet

G‐rich nucleic acid sequences with the potential to form G‐quadruplex structures are common in biologically important regions. Most of these sequences are present with their complementary strands, so the development of a sensitive biosensor to distinguish G‐quadruplex and duplex structures and to determine the competitive ability of quadruplex to duplex structures has received a great deal of attention. In this work, the interactions between two triphenylmethane dyes (malachite green (MG) and crystal violet (CV)) and G‐quadruplex, duplex, or single‐stranded DNAs were studied by fluorescence spectroscopy and energy‐transfer fluorescence spectroscopy. Good discrimination between quadruplexes and duplex or single‐stranded DNAs can be achieved by using the fluorescence spectrum of CV or the energy‐transfer fluorescence spectra of CV and MG. In addition, by using energy‐transfer fluorescence titrations of CV with G‐quadruplexes, the binding‐stoichiometry ratios of CV to G‐quadruplexes can be determined. By using the fluorescence titrations of G‐quadruplex–CV complexes with C‐rich complementary strands, the fraction of G‐rich oligonucleotide that engages in G‐quadruplex structures in the presence of the complementary sequence can be measured. This study may provide a simple method for discrimination between quadruplexes and duplex or single‐stranded DNAs and for measuring G‐quadruplex percentages in the presence of the complementary C‐rich sequences.     

Examination of the Effect of the Annealing Cation on Higher Order Structures Containing Guanine or Isoguanine Repeats

Isoguanine (2‐oxo‐6‐amino‐guanine), a natural but non‐standard base, exhibits unique self‐association properties compared to its isomer, guanine, and results in formation of different higher order DNA structures. In this work, the higher order structures formed by oligonucleotides containing guanine repeats or isoguanine repeats after annealing in solutions containing various cations are evaluated by electrospray ionization mass spectrometry (ESI‐MS) and circular dichroism (CD) spectroscopy. The guanine‐containing strand (G9) consistently formed quadruplexes upon annealing, whereas the isoguanine strand (Ig9) formed both pentaplexes and quadruplexes depending on the annealing cation. Quadruplex formation with G9 showed some dependence on the identity of the cation present during annealing with high relative quadruplex formation detected with six of ten cations. Analogous annealing experiments with Ig9 resulted in complex formation with all ten cations, and the majority of the resulting complexes were pentaplexes. CD results indicated most of the original complexes survived the desalting process necessary for ESI‐MS analysis. In addition, several complexes, especially the pentaplexes, were found to be capable of cation exchange with ammonium ions. Ab initio calculations were conducted for isoguanine tetrads and pentads coordinated with all ten cations to predict the most energetically stable structures of the complexes in the gas phase. The observed preference of forming quadruplexes versus pentaplexes as a function of the coordinated cation can be interpreted by the calculated reaction energies of both the tetrads and pentads in combination with the distortion energies of tetrads.     

Ion‐Responsive Hemin–G‐Quadruplexes for Switchable DNAzyme and Enzyme Functions

Programmed nucleic acid sequences undergo K⁺ ion‐induced self‐assembly into G‐quadruplexes and separation of the supramolecular structures by the elimination of K⁺ ions by crown ether or cryptand ion‐receptors. This process allows the switchable formation and dissociation of the respective G‐quadruplexes. The different G‐quadruplex structures bind hemin, and the resulting hemin–G‐quadruplex structures reveal horseradish peroxidase DNAzyme catalytic activities. The following K⁺ ion/receptor switchable systems are described: 1) The K⁺‐induced self‐assembly of the Mg²⁺‐dependent DNAzyme subunits into a catalytic nanostructure using the assembly of G‐quadruplexes as bridging unit. 2) The K⁺‐induced stabilization of the anti‐thrombin G‐quadruplex nanostructure that inhibits the hydrolytic functions of thrombin. 3) The K⁺‐induced opening of DNA tweezers through the stabilization of G‐quadruplexes on the “tweezers’ arms" and the release of a strand bridging the tweezers into a closed structure. In all of the systems reversible, switchable, functions are demonstrated. For all systems two different signals are used to follow the switchable functions (fluorescence and the catalytic functions of the derived hemin–G‐quadruplex DNAzyme).     

Metal‐Ion‐Triggered Exonuclease III Activity for the Construction of DNA Colorimetric Logic Gates

The logic system is obtained by using a series of double‐stranded (ds) DNA templates with mismatched base pairs (T–T or C–C) and ion‐modulated exonuclease III (Exo III) activity, in which the Exo III cofactors, Hg²⁺ and Ag⁺ ions, are used as inputs for the activation of the respective scission of Exo III based on the formation of T–Hg²⁺–T or C–Ag⁺–C base pairs. Additionally, two kinds of signal probes are utilized to transduce the logic operations. One is the two split G‐rich DNA strands that are used to design the OR, AND, INHIBIT, and XOR gates, whereas the other is the self‐assembled split G‐quadruplex structure to construct NOR, NAND, IMPLICATION, and XNOR operations based on DNA hybridization and strand displacement. In the presence of hemin, the split G‐quadruplex biocatalyzes the formation of a colored product, which is an output signal for the different logic gates. Thus, we have constructed a complete set of colorimetric DNA logic gates based on the Exo III and split G‐quadruplex for the first time. In addition, we are able to effortlessly recognize the logic output signals by the naked eye and their simplicity and cost‐effective design is the most apparent feature for the logic gates developed in this work.     

Neutral and Negatively Charged Phosphate Modifications Altering Thermal Stability,Kinetics of Formation and Monovalent Ion Dependence of DNA G‐Quadruplexes

The effect of phosphate group modifications on formation and properties of G‐quadruplexes (G4s) has not been investigated in detail. Here, we evaluated the structural, thermodynamic and kinetic properties of the parallel G‐quadruplexes formed by oligodeoxynucleotides d(G₄T), d(TG₄T) and d(TG₅T), in which all phosphates were replaced with N‐methanesulfonyl (mesyl) phosphoramidate or phosphoryl guanidine groups resulting in either negatively charged or neutral DNA sequences, respectively. We established that all modified sequences were able to form G‐quadruplexes of parallel topology; however, the presence of modifications led to a decrease in thermal stability relative to unmodified G4s. In contrast to negatively charged G4s, assembly of neutral G4 DNA species was faster in the presence of sodium ions than potassium ions, and was independent of the salt concentration used. Formation of mixed G4s composed of both native and neutral G‐rich strands has been detected using native gel electrophoresis, size‐exclusion chromatography and ESI‐MS. In summary, our results indicate that the phosphate modifications studied are compatible with G‐quadruplex formation, which could be used for the design of biologically active compounds.     

Monomer–Dimer Equilibrium for the 5′–5′ Stacking of Propeller‐Type Parallel‐Stranded G‐Quadruplexes: NMR Structural Study

Guanine‐rich sequence motifs, which contain tracts of three consecutive guanines connected by single non‐guanine nucleotides, are abundant in the human genome and can form a robust G‐quadruplex structure with high stability. Herein, by using NMR spectroscopy, we investigate the equilibrium between monomeric and 5′–5′ stacked dimeric propeller‐type G‐quadruplexes that are formed by DNA sequences containing GGGT motifs. We show that the monomer–dimer equilibrium depends on a number of parameters, including the DNA concentration, DNA flanking sequences, the concentration and type of cations, and the temperature. We report on the high‐definition structure of a simple monomeric G‐quadruplex containing three single‐residue loops, which could serve as a reference for propeller‐type G‐quadruplex structures in solution.     

Solution NMR Structure of a Ligand/Hybrid‐2‐G‐Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding

Telomeric G‐quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G‐quadruplex that adopts the biologically relevant hybrid‐2 conformation in a ligand‐bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G‐quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G‐quadruplex. The ligand is sandwiched between one terminal G‐tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G‐quadruplex structure as observed for other G‐quadruplexes in different conformations, invalidating simple docking approaches to ligand–G‐quadruplex structure determination.     

Fluorescent Bioprobes: Structural Matching in the Docking Processes of Aggregation‐Induced Emission Fluorogens on DNA Surfaces

Whereas most conventional DNA probes are flat disklike aromatic molecules, we explored the possibility of developing quadruplex sensors with nonplanar conformations, in particular, the propeller‐shaped tetraphenylethene (TPE) salts with aggregation‐induced emission (AIE) characteristics. 1,1,2,2‐Tetrakis[4‐(2‐triethylammonioethoxy)phenyl]ethene tetrabromide (TPE‐ 1 ) was found to show a specific affinity to a particular quadruplex structure formed by a human telomeric DNA strand in the presence of K⁺ ions, as indicated by the enhanced and bathochromically shifted emission of the AIE fluorogen. Steady‐state and time‐resolved spectral analyses revealed that the specific binding stems from a structural matching between the AIE fluorogen and the DNA strand in the folding process. Computational modeling suggests that the AIE molecule docks on the grooves of the quadruplex surface with the aid of electrostatic attraction. The binding preference of TPE‐ 1 enables it to serve as a bioprobe for direct monitoring of cation‐driven conformational transitions between the quadruplexes of various conformations, a job unachievable by the traditional G‐quadruplex biosensors. Methyl thiazolyl tetrazolium (MTT) assays reveal that TPE‐ 1 is cytocompatible, posing no toxicity to living cells.     

The Effect of DNA Sequence Directionality on G‐Quadruplex Folding

Sequence inversion in G‐rich DNA from 5′→3′ to 3′→5′ exerts a substantial effect on the number of structures formed, while the type of G‐quadruplex fold is in fact determined by the presence of K⁺ or Na⁺ ions. The melting temperatures of G‐quadruplexes adopted by oligonucleotides with sequences in the 5′→3′ direction are higher than those of their 3′→5′ counterparts with both KCl and NaCl. CD, UV, and NMR spectroscopy demonstrates the importance of primary sequence for the structural diversity of G‐quadruplexes. The changes introduced by mere sequence reversal of the G‐rich DNA segment have a substantial impact on the polymorphic nature of the resulting G‐quadruplexes and their potential physiological roles. The insights resulting from this study should enable extension of the empirical rules for the prediction of G‐quadruplex topology.     

A Flexible Terpyridine Derivative Interacts Specifically with G‐Quadruplexes

G‐quadruplexes formed by nucleic acids are implicated in pathologies ranging from cancers to neurodegenerative diseases. We evaluated interactions of 29 bi‐ and terpyridine derivatives with G‐quadruplexes and duplexes. FRET‐melting, circular dichroism, and ¹H NMR spectroscopy showed that one terpyridine derivative interacted very selectively with G‐quadruplexes. This G‐quadruplex ligand inhibited helicase activity and should influence G‐quadruplex‐related biological processes.     

Photo‐Cross‐Linking Probes for Trapping G‐Quadruplex DNA

We have developed a straightforward synthetic pathway to a set of six photoactivatable G‐quadruplex ligands with a validated G4‐binding motif (the bisquinolinium pyridodicarboxamide PDC‐360A) tethered through various spacers to two different photo‐cross‐linking groups: benzophenone and an aryl azide. The high quadruplex‐versus‐duplex selectivity of the PDC core was retained in the new derivatives and resulted in selective alkylation of two well‐known G‐quadruplexes (human telomeric G4 and oncogene promoter c‐myc G4) under conditions of harsh competition. The presence of two structurally different photoactivatable functions allowed the selective alkylation of G‐quadruplex structures at specific nucleobases and irreversible G4 binding. The topology and sequence of the quadruplex matrix appear to influence strongly the alkylation profile, which differs for the telomeric and c‐myc quadruplexes. The new compounds are photoactive in cells and thus provide new tools for studying G4 biology.     

Effect of the Ancillary Ligands on the Spectral Properties and G‐Quadruplexes DNA Binding Behavior: A Combined Experimental and Theoretical Study

In an effort to explore the effect of ancillary ligands on the spectral properties and overall G‐quadruplex DNA binding behavior, two new ruthenium(II) complexes [Ru(phen)₂(dppzi)]²⁺ ( 1 ) and [Ru(dmp)₂(dppzi)]²⁺ ( 2 ) (phen=1,10‐phenanthroline, dmp=2,9‐dimethyl‐1,10‐phenanthroline, dppzi=dipyrido[3,2‐a:2′,3′‐c]phenazine‐10,11‐imidazole) were prepared. Complex 1 can emit luminescence in the absence and presence of G‐quadruplexes DNA. However, with ?CH₃ substituent on the 2‐ and 9‐positions of the phen ancillary ligand, no detectable luminescence is observed for complex 2 in any organic solvent or in the absence and/or presence of G‐quadruplex DNA. Experimental and molecular docking studies indicated that both complexes interacted with the human telomeric repeat AG3(T2AG3)3 (22AG) G‐quadruplex with the stoichiometric ratio of 1:1, but the two complexes showed different G‐quadruplex DNA binding affinity. Complex 1 binds to the G‐quadruplexes DNA more tightly than complex 2 does. Our results demonstrate that methyl groups on the phen ancillary ligand significantly affect the spectral properties and the overall DNA binding behavior of the complexes. Such difference in spectral properties and DNA binding affinities of these two complexes can be reasonably explained by DFT/TD‐DFT calculations. This work provides guidance not only on exploring the G‐quadruplexes DNA binding behavior of complexes, but also understanding the unique luminescence mechanism.     

Pyridyl‐Substituted Corrole Isomers: Synthesis and their Regulation to G‐quadruplex Structures

G‐quadruplex DNA plays an important role in the potential therapeutic target for the design and development of anticancer drugs. As various G‐quadruplex sequences in the promoter regions or telomeres can form different secondary structural modes and display a diversity of biology functions, variant G‐quadruplex interactive agents may be necessary to cure different disease by differentiating variant types of G‐quadruplexes. We synthesize five cationic methylpyridylium corroles and compare the interactions of corroles with different types of G‐quadruplexes such as cmyc, htelo, and bcl2 by using surface plasmon resonance. Because of the importance of human telomere G‐quadruplex DNA, we focus on the biological properties of the interactions between human telomere G‐quadruplex DNA and corrole isomers using CD, T_m, PCR‐stop (PCR= polymerase chain reaction), and polymerase‐stop assay, which demonstrate the excellent ability of the corrole to induce and stabilize the G‐quadruplex. This study provides the first experimental insight into how selectivity might be achieved for different G‐quadruplexes by a single group of methylpyridylium corrole isomers that may be optimized for potential selective cancer therapy.     

Photocrosslinking of G‐Quadruplex‐Forming Sequences found in Human Promoters

While is it well known that human telomeric DNA sequences can adopt G‐quadruplex structures, some promoters sequences have also been found to form G‐quadruplexes, and over 40% of promoters contain putative G‐quadruplex‐forming sequences. Because UV light has been shown to crosslink human telomeric G‐quadruplexes by cyclobutane pyrimidine dimer (CPD) formation between T's on adjacent loops, UV light might also be able to photocrosslink G‐quadruplexes in promoters. To investigate this possibility, 15 potentially UV‐crosslinkable G‐quadruplex‐forming sequences found in a search of human DNA promoters were UVB irradiated in vitro, and three were confirmed to have formed nonadjacent CPDs by mass spectrometry. In addition to nonadjacent T=T CPDs found in human telomeric DNA, a nonadjacent T=U CPD was discovered that presumably arose from deamination of a nonadjacent T=C CPD. Analysis of the three sequences by circular dichroism, melting temperature analysis and chemical footprinting confirmed the presence of G‐quadruplexes that could explain the formation of the nonadjacent CPDs. The formation of nonadjacent CPDs from the sequences in vitro suggests that they might be useful probes for the presence of non‐B DNA structures, such as G‐quadruplexes, in vivo, and if they were to form in vivo, might also have significant biological consequences.     

Oligonucleotide Analogues with Integrated Bases and Backbone (ONIB). Part 31

The self‐complementary guanosine‐ and cytidine‐derived aminomethylene‐linked C*[n ]G dinucleoside 9 was synthesized by reductive amination of aldehyde 3 with an iminophosphorane derived from azide 7 . Deacylation of 9 gave the isopropylidene‐protected dinucleoside 10 . The sequence‐isomeric G*[n ]C dinucleoside 11 was similarly prepared from aldehyde 8 and azide 5 , and deacylated to 12 . The association of 10 and 12 in CHCl₃ or in CHCl₃/DMSO mixtures, and the structure of the associates were studied by ¹H‐NMR, ESI‐MS, CD, and vapor pressure osmometry (VPO). Broad ¹H‐NMR signals of dinucleosides 10 and 12 evidence an equilibrium between duplexes and quadruplexes (Hoogsteen base pairing between the Watson? Crick base‐paired duplexes). The quadruplex dominates for the G*[n ]C dinucleoside 12 between ?50° and room temperature. The sequence‐isomeric C*[n ]G 10 forms mostly only a cyclic duplex in CDCl₃ and in CDCl₃/(D₆)DMSO 9 : 1.     

Interaction of Metal Complexes with G‐Quadruplex DNA

Guanine‐rich sequences of DNA can assemble into tetrastranded structures known as G‐quadruplexes. It has been suggested that these secondary DNA structures could be involved in the regulation of several key biological processes. In the human genome, guanine‐rich sequences with the potential to form G‐quadruplexes exist in the telomere as well as in promoter regions of certain oncogenes. The identification of these sequences as novel targets for the development of anticancer drugs has sparked great interest in the design of molecules that can interact with quadruplex DNA. While most reported quadruplex DNA binders are based on purely organic templates, numerous metal complexes have more recently been shown to interact effectively with this DNA secondary structure. This Review provides an overview of the important roles that metal complexes can play as quadruplex DNA binding molecules, highlighting the unique properties metals can confer to these molecules.     

An ESI‐MS and NMR Study of the Self‐Assembly of Guanosine Derivatives

The self‐assembly of guanosine (G) derivatives in the presence of alkali‐metal ions gives octameric or polymeric aggregates composed of stacked G quartets. This process is studied for some lipophilic G derivatives by means of ESI‐MS. The ESI‐MS results are discussed in the light of complementary information obtained from NMR and SANS (small‐angle neutron scattering) studies. ESI‐MS gives an excellent picture of the self‐assembly process and gives new information on the effect of different cations and anions on the dimensions of the assembled species, information that could not have been obtained with SANS and NMR alone.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.     

Electrospray‐Ionization Mass Spectrometry for Screening the Specificity and Stability of Single‐Stranded‐DNA Templated Self‐Assemblies

Supramolecular complexes consisting of a single‐stranded oligothymine ( dTn ) as the host template and an array of guest molecules equipped with a complementary diaminotriazine hydrogen‐bonding unit have been studied with electrospray‐ionization mass spectrometry (ESI‐MS). In this hybrid construct, a supramolecular stack of guest molecules is hydrogen bonded to dTn . By changing the hydrogen‐bonding motif of the DNA host template or the guest molecules, selective hydrogen bonding was proven. We were able to detect single‐stranded‐DNA (ssDNA)–guest complexes for strands with lengths of up to 20 bases, in which the highest complex mass detected was 15 kDa; these complexes constitute 20‐component self‐assembled objects. Gas‐phase breakdown experiments on single‐ and multiple‐guest–DNA assemblies gave qualitative information on the fragmentation pathways and the relative complex stabilities. We found that the guest molecules are removed from the template one by one in a highly controlled way. The stabilities of the complexes depend mainly on the molecular weight of the guest molecules, a fact suggesting that the complexes collapse in the gas phase. By mixing two different guests with the ssDNA template, a multicomponent dynamic library can be created. Our results demonstrate that ESI‐MS is a powerful tool to analyze supramolecular ssDNA complexes in great detail.