Higher‐Order Human Telomeric G‐Quadruplex DNA Metalloenzymes Enhance Enantioselectivity in the Diels–Alder Reaction

Short human telomeric (HT) DNA sequences form single G‐quadruplex (G₄) units and exhibit structure‐based stereocontrol for a series of reactions. However, for more biologically relevant higher‐order HT G₄‐DNAs (beyond a single G₄ unit), the catalytic performances are unknown. Here, we found that higher‐order HT G₄‐DNA copper metalloenzymes (two or three G₄ units) afford remarkably higher enantioselectivity (>90 % ee) and a five‐ to sixfold rate increase, compared to a single G₄ unit, for the Diels–Alder reaction. Electron paramagnetic resonance (EPR) and enzymatic kinetic studies revealed that the distinct catalytic function between single and higher‐order G₄‐DNA copper metalloenzymes can be attributed to different Cu^II coordination environments and substrate specificity. Our finding suggests that, like protein enzymes and ribozymes, higher‐order structural organization is crucial for G₄‐DNA‐based catalysis.     

Rational Design,Synthesis, Biophysical and Antiproliferative Evaluation of Fluorenone Derivatives with DNA G‐Quadruplex Binding Properties

Molecular modeling studies carried out with experimental DNA models with the sequence d[AG₃(T₂AG₃)₃] suggest that the introduction of a net positive charge onto the side chain of a series of fluorenone carboxamides can improve G‐quadruplex binding. The terminal morpholino moiety was replaced with a novel N‐methylmorpholinium cation starting from two 4‐carboxamide compounds. A different substitution on the fluorenone ring was also investigated and submitted to the same quaternarization process. All compounds were analyzed for their DNA binding properties by competition dialysis methods. In vitro antiproliferative tests were carried out against two different tumor cell lines. Docking experiments were conducted by including all four known human repeat unit G‐quadruplex DNA sequences (27 experimentally determined conformations) against the most active fluorenone derivatives. The results of theoretical, biophysical, and in vitro experiments indicate two novel derivatives as lead compounds for the development of a new generation of G‐quadruplex ligands with greater potency and selectivity.     

Molecular Recognition of Parallel DNA Quadruplex d(TTAGGGT)4 by Mitoxantrone: Binding with 1:2 Stoichiometry Leading to Thermal Stabilization and Telomerase Inhibition

The interaction of the anthraquinone derivative mitoxantrone, a semisynthetic anti‐cancer drug with two non‐planar side chains, with heptamer G‐quadruplex d(TTAGGGT)₄, which contains the human telomere DNA sequence, was evaluated by differential scanning calorimetry, fluorescence Job plotting, absorption, and NMR and CD spectroscopy. Binding led to thermal stabilization of DNA (ΔT_m=13–20 °C). The spectra revealed that two mitoxantrone molecules bind externally at two sites of the DNA quadruplex as monomers, by partial insertion of the chromophore and side‐chain interaction at the grooves. The inhibition of telomerase (IC₅₀=2 μm), as determined by a TRAP assay, can be attributed to thermal stabilization of the DNA quadruplex because of the interactions with mitoxantrone. The studies revealed highly specific molecular recognition between a ligand and a parallel‐stranded G‐quadruplex; this might serve as a platform for the rational design of new drugs.     

Differential Inhibitory Activities and Stabilisation of DNA Aptamers against the SARS Coronavirus Helicase

The helicase from severe acute respiratory syndrome coronavirus (SARS‐CoV) possesses NTPase, duplex RNA/DNA‐unwinding and RNA‐capping activities that are essential for viral replication and proliferation. Here, we have isolated DNA aptamers against the SARS‐CoV helicase from a combinatorial DNA library. These aptamers show two distinct classes of secondary structure, G‐quadruplex and non‐G‐quadruplex, as shown by circular dichroism and gel electrophoresis. All of the aptamers that were selected stimulated ATPase activity of the SARS‐CoV helicase with low‐nanomolar apparent K_m values. Intriguingly, only the non‐G‐quadruplex aptamers showed specific inhibition of helicase activities, whereas the G‐quadruplex aptamers did not inhibit helicase activities. The non‐G‐quadruplex aptamer with the strongest inhibitory potency was modified at the 3′‐end with biotin or inverted thymidine, and the modification increased its stability in serum, particularly for the inverted thymidine modification. Structural diversity in selection coupled to post‐selection stabilisation has provided new insights into the aptamers that were selected for a helicase target. These aptamers are being further developed to inhibit SARS‐CoV replication.     

Triplex‐Forming Twisted Intercalating Nucleic Acids (TINAs): Design Rules,Stabilization of Antiparallel DNA Triplexes and Inhibition of G‐Quartet‐Dependent Self‐Association

The majority of studies on DNA triple helices have been focused on pH‐sensitive parallel triplexes with Hoogsteen CT‐containing third strands that require protonation of cytosines. Reverse Hoogsteen GT/GA‐containing antiparallel triplex‐forming oligonucleotides (TFOs) do not require an acidic pH but their applicability in triplex technology is limited because of their tendency to form undesired highly stable aggregates such as G‐quadruplexes. In this study, G‐rich oligonucleotides containing 2–4 insertions of twisted intercalating nucleic acid (TINA) monomers are demonstrated to disrupt the formation of G‐quadruplexes and form stable antiparallel triplexes with target DNA duplexes. The structure of TINA‐incorporated oligonucleotides was optimized, the rules of their design were established and the optimal triplex‐forming oligonucleotides were selected. These oligonucleotides show high affinity towards a 16 bp homopurine model sequence from the HIV‐1 genome; dissociation constants as low as 160 nM are observed whereas the unmodified TFO does not show any triplex formation and instead forms an intermolecular G‐quadruplex with T_m exceeding 90 °C in the presence of 50 mM NaCl. Here we present a set of rules that help to reach the full potential of TINA‐TFOs and demonstrate the effect of TINA on the formation and stability of triple helical DNA.     

Efficient Inhibition of Telomerase by Nickel–Salophen Complexes

Four nickel(II)–salophen complexes containing alkyl‐imidazolium chains connected at the ortho or meta positions were prepared: N,N′‐bis(2‐hydroxy‐4‐methyl‐3H‐imidazol‐1‐iumbenzylideneamino)phenylenediamine ( 1 ), N,N′‐bis(2‐hydroxy‐3‐methyl‐3H‐imidazol‐1‐iumbenzylideneamino)phenylenediamine ( 2 ), N,N′‐bis(2‐hydroxy‐3‐methyl‐3H‐imidazol‐1‐iumbenzylideneamino)methyl‐3H‐imidazol‐1‐iumphenylenediamine ( 3 ), and N,N′‐bis(2‐hydroxy‐4‐methyl‐3H‐imidazol‐1‐iumbenzylideneamino)methyl‐3H‐imidazol‐1‐iumphenylenediamine ( 4 ). They protect G‐quadruplex DNA (G₄‐DNA) against thermal denaturation and show K_A values in the range of 7.4×10⁵ to 4×10⁷ m ^?1 for G₄‐DNA models. Complex 4 exhibits an IC₅₀ value of 70 nm for telomerase inhibition.     

Effect of ATRX and G‐Quadruplex Formation by the VNTR Sequence on α‐Globin Gene Expression

ATR‐X (α‐thalassemia/mental retardation X‐linked) syndrome is caused by mutations in chromatin remodeler ATRX. ATRX can bind the variable number of tandem repeats (VNTR) sequence in the promoter region of the α‐globin gene cluster. The VNTR sequence, which contains the potential G‐quadruplex‐forming sequence CGC(GGGGCGGGG)_n, is involved in the downregulation of α‐globin expression. We investigated G‐quadruplex and i‐motif formation in single‐stranded DNA and long double‐stranded DNA. The promoter region without the VNTR sequence showed approximately twofold higher luciferase activity than the promoter region harboring the VNTR sequence. G‐quadruplex stabilizers hemin and TMPyP4 reduced the luciferase activity, whereas expression of ATRX led to a recovery in reporter activity. Our results demonstrate that stable G‐quadruplex formation by the VNTR sequence downregulates the expression of α‐globin genes and that ATRX might bind to and resolve the G‐quadruplex.     

Supramolecular Polymorphism of (G4C2)n Repeats Associated with ALS and FTD

Guanine-rich DNA sequences self-assemble into highly stable fourfold structures known as DNA-quadruplexes (or G-quadruplexes). G-quadruplexes have furthermore the tendency to associate into one-dimensional supramolecular aggregates termed G-wires. We studied the formation of G-wires in solutions of the sequences d(G₄C₂)_n with n = 1, 2, and 4. The d(G₄C₂)_n repeats, which are associated with some fatal neurological disorders, especially amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), represent a challenging research topic due to their extensive structural polymorphism. We used dynamic light scattering (DLS) to measure translational diffusion coefficients and consequently resolve the length of the larger aggregates formed in solution. We found that all three sequences assemble into longer structures than previously reported. The d(G₄C₂) formed extremely long G-wires with lengths beyond 80 nm. The d(G₄C₂)₂ formed a relatively short stacked dimeric quadruplex, while d(G₄C₂)₄ formed multimers corresponding to seven stacked intramolecular quadruplexes. Profound differences between the multimerization properties of the investigated sequences were also confirmed by the AFM imaging of surface films. We propose that π-π stacking of the basic G-quadruplex units plays a vital role in the multimerization mechanism, which might be relevant for transformation from the regular medium-length to disease-related long d(G₄C₂)_n repeats.     

Development of an Efficient G‐Quadruplex‐Stabilised Thrombin‐Binding Aptamer Containing a Three‐Carbon Spacer Molecule

The thrombin‐binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G‐quadruplex‐forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three‐carbon spacer (spacer‐C₃), unlocked nucleic acid (UNA) and 3′‐amino‐modified UNA (amino‐UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G‐quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer‐C₃ introduction at the T7 loop position (TBA‐SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA‐SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.     

Fluorescent Probes for G‐Quadruplex Structures

Mounting evidence supports the presence of biologically relevant G‐quadruplexes in single‐cell organisms, but the existence of endogenous G‐quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure–function relationships of nucleic acids. In this review, we present progress towards the direct detection of G‐quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell‐permeable probes that selectively bind to G‐quadruplex structures with high affinity, these same probes can induce G‐quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as “internal” fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G‐quadruplex, single‐stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G‐quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.     

Targeting DNA G‐Quadruplexes with Helical Small Molecules

We previously identified quinoline‐based oligoamide helical foldamers and a trimeric macrocycle as selective ligands of DNA quadruplexes. Their helical structures might permit targeting of the backbone loops and grooves of G‐quadruplexes instead of the G‐tetrads. Given the vast array of morphologies G‐quadruplex structures can adopt, this might be a way to achieve sequence selective binding. Here, we describe the design and synthesis of molecules based on macrocyclic and helically folded oligoamides. We tested their ability to interact with the human telomeric G‐quadruplex and an array of promoter G‐quadruplexes by using FRET melting assay and single‐molecule FRET. Our results show that they constitute very potent ligands—comparable to the best so far reported. Their modes of interaction differ from those of traditional tetrad binders, thus opening avenues for the development of molecules specific for certain G‐quadruplex conformations.     

G-Quadruplex Formation in a Putative Coding Region of White Spot Syndrome Virus: Structural and Thermodynamic Aspects

White spot disease (WSD) is one of the most devastating viral infections of crustaceans caused by the white spot syndrome virus (WSSV). A conserved sequence WSSV131 in the DNA genome of WSSV was found to fold into a polymorphic G-quadruplex structure. Supported by two mutant sequences with single G→T substitutions in the third G₄ tract of WSSV131, circular dichroism and NMR spectroscopic analyses demonstrate folding of the wild-type sequence into a three-tetrad parallel topology comprising three propeller loops with a major 1 : 3 : 1 and a minor 1 : 2 : 2 loop length arrangement. A thermodynamic analysis of quadruplex formation by differential scanning calorimetry (DSC) indicates a thermodynamically more stable 1 : 3 : 1 loop isomer. DSC also revealed the formation of additional highly stable multimeric species with populations depending on potassium ion concentration.     

Aromatic Core Extension in the Series of N‐Cyclic Bay‐Substituted Perylene G‐Quadruplex Ligands: Increased Telomere Damage,Antitumor Activity,and Strong Selectivity for Neoplastic over Healthy Cells

Based on previous work on both perylene and coronene derivatives as G‐quadruplex binders, a novel chimeric compound was designed: N,N′‐bis[2‐(1‐piperidino)‐ethyl]‐1‐(1‐piperidinyl)‐6‐[2‐(1‐piperidino)‐ethyl]‐benzo[ghi]perylene‐3,4:9,10‐tetracarboxylic diimide (EMICORON), having one piperidinyl group bound to the perylene bay area (positions 1, 12 and 6, 7 of the aromatic core), sufficient to guarantee good selectivity, and an extended aromatic core able to increase the stacking interactions with the terminal tetrad of the G‐quadruplex. The obtained “chimera” molecule, EMICORON, rapidly triggers extensive DNA damage of telomeres, associated with the delocalization of telomeric protein protection of telomeres 1 (POT1), and efficiently limits the growth of both telomerase‐positive and ‐negative tumor cells. Notably, the biological effects of EMICORON are more potent than those of the previously described perylene derivative (PPL3C), and more interestingly, EMICORON appears to be detrimental to transformed and tumor cells, while normal fibroblasts expressing telomerase remain unaffected. These results identify a new promising G‐quadruplex ligand, structurally and biologically similar on one side to coronene and on the other side to a bay‐monosubstituted perylene, that warrants further studies.     

Duplex Healing of Selectively Thiolated Guanosine Mismatches through a Cd2+ Chemical Stimulus

The on‐column selective conversion of guanosine to thioguanosine (tG) yields modified oligomers that exhibit destabilisation over the fully complementary duplex. Restoration to a stabilised duplex is induced through thio‐directed Cd²⁺ coordination; a route for healing DNA damage. Short oligomers are G‐specifically thiolated through a modified on‐column protocol without the need for costly thioguanosine phosphoramidites. Addition of Cd²⁺ ions to a duplex containing a highly disrupted tG central mismatch sequence, 3′‐A₆tG₄T₆‐5′, suggests a (tG)₈Cd₂ central coordination regime, resulting in increased base stacking and duplex stability. Equilibrium molecular dynamic calculations support the hypothesis of metal‐induced healing of the thiolated duplex. The 2 nm displacement of the central tG mismatched region is dramatically reduced after the addition of a chemical stimuli, Cd²⁺ ions, returning to a minimized fluctuational state comparable to the unmodified fully complementary oligomer.