Multivalent Interactions between an Aromatic Helical Foldamer and a DNA G‐Quadruplex in the Solid State

Quinoline‐based oligoamide foldamers have been identified as a potent class of ligands for G‐quadruplex DNA. Their helical structure is thought to target G‐quadruplex loops or grooves and not G‐tetrads. We report a co‐crystal structure of the antiparallel hairpin dimeric DNA G‐quadruplex (G₄T₄G₄)₂ with tetramer 1 —a helically folded oligo‐quinolinecarboxamide bearing cationic side chains—that is consistent with this hypothesis. Multivalent foldamer–DNA interactions that modify the packing of (G₄T₄G₄)₂ in the solid state are observed.     

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.     

G‐Quadruplexes as Tools for Synthetic Biology

With the potential to engineer biological systems, synthetic biology is an emerging field that combines various disciplines of sciences. It encompasses combinations of DNA, RNA and protein modules for constructing desired systems and the “rewiring” of existing signalling networks. Despite recent advances, this field still lags behind in the artificial reconstruction of cellular processes, and thus demands new modules and switches to create “genetic circuits”. The widely characterised noncanonical nucleic acid secondary structures, G‐quadruplexes are promising candidates to be used as biological modules in synthetic biology. Structural plasticity and functional versatility are significant G‐quadruplex traits for its integration into a biological system and for diverse applications in synthetic circuits.     

Looking for Efficient G‐Quadruplex Ligands: Evidence for Selective Stabilizing Properties and Telomere Damage by Drug‐Like Molecules

There is currently significant interest in the development of G‐quadruplex‐interactive compounds, given the relationship between the ability to stabilize these non‐canonical DNA structures and anticancer activity. In this study, a set of biophysical assays was applied to evaluate the binding of six drug‐like ligands to DNA G‐quadruplexes with different folding topologies. Interestingly, two of the investigated ligands showed selective G‐quadruplex‐stabilizing properties and biological activity. These compounds may represent useful leads for the development of more potent and selective ligands.     

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.     

Optimizing the Kinetics and Thermodynamics of DNA i‐Motif Folding

Under slightly acidic conditions, single cytidine‐rich DNA strands can form four‐stranded structures called i‐motifs. The stability of the i‐motif structure is based on the intercalation of hemiprotonated C–C⁺ base pairs. In addition, the stability of these structures is influenced by pH, temperature, salt concentration, number of cytidines per C‐rich stretch, and length of sequence; it also depends on the nucleotides in the connecting loop regions. Here, we investigated the influence of the loop nucleotides on i‐motif stability, structure, and kinetics of folding, in five structures with the same loop‐size but different adenosine and thymidine residues within the loop. The stabilities of the i‐motif structures were determined by CD melting, and structure and kinetics of folding were studied by static and time‐resolved NMR experiments.     

Structurally Diverse Polyamines: Solid‐Phase Synthesis and Interaction with DNA

A versatile solid‐phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher‐order structures of DNA.     

5‐Hydroxymethyl‐2′‐Deoxyuridine Residues in the Thrombin Binding Aptamer: Investigating Anticoagulant Activity by Making a Tiny Chemical Modification

We report an investigation into analogues of the thrombin binding aptamer (TBA). Individual thymidines were replaced by the unusual residue 5‐hydroxymethyl‐2′‐deoxyuridine (hmU). This differs from the canonical thymidine by a hydroxyl group on the 5‐methyl group. NMR and CD data clearly indicate that all TBA derivatives retain the ability to fold into the “chair‐like” quadruplex structure. The presence of the hmU residue does not significantly affect the thermal stability of the modified aptamers compared to the parent, except for analogue H9 , which showed a marked increase in melting temperature. Although all TBA analogues showed decreased affinities to thrombin, H3 , H7 , and H9 proved to have improved anticoagulant activities. Our data open up the possibility to enhance TBA biological properties, simply by introducing small chemical modifications.     

Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G‐Quadruplexes

We have developed fluorescent protein probes specific for parallel G‐quadruplexes by attaching cyan fluorescent protein to the G‐quadruplex‐binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G‐quadruplexes was characterized. The selective recognition and discrimination of G‐quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging.     

Reduction‐Responsive Guanine Incorporated into G‐Quadruplex‐Forming DNA

Stimulus‐responsive biomolecules are attractive targets to understand biomolecule behaviour as well as to explore their therapeutic and diagnostic applications. We demonstrate that a reduction‐responsive cleavable group (chemically caged unit) introduced into the guanine ring enables modulation of the secondary structure transition of an oligonucleotide in a reduction‐responsive and traceless manner leaving the unmodified oligonucleotide of interest. This simple but robust strategy could yield a variety of stimuli‐responsive oligonucleotides.     

Cellular Internalization of Water‐Soluble Helical Aromatic Amide Foldamers

The intracellular transport of drugs and therapeutics represents one of the most exciting and challenging areas at the interface of chemistry, biology, and medicine. Most of the effort in this field so far has been devoted to the development of peptide‐based delivery systems that can translocate therapeutic agents into their intracellular targets. More recently, the use of bioinspired non‐natural foldamers has resulted in the successful delivery of cargo molecules, which possess a wide range of sizes and physicochemical properties across the cell membrane. We report herein the synthesis of aromatic amide foldamers and their biological evaluation as cell‐penetrating agents. By using a well‐established synthetic route, a series of fluorescein‐labeled cationic aryl amide conjugates has been constructed, and their cellular uptake into various human cell lines has been analyzed by flow cytometry and fluorescence microscopy. The assays revealed that longer oligomers achieve greater cellular translocation, with octamer Q8 proving to be a remarkable vehicle for all three cell lines. Biological studies have also indicated that these helices are biocompatible, thus showing promise in their application as cell‐penetrating agents and as vehicles to deliver biologically active molecules into cells.