Photoactivatable V-Shaped Bifunctional Quinone Methide Precursors as a New Class of Selective G-quadruplex Alkylating Agents

Combining the selectivity of G-quadruplex (G4) ligands with the spatial and temporal control of photochemistry is an emerging strategy to elucidate the biological relevance of these structures. In this work, we developed six novel V-shaped G4 ligands that can, upon irradiation, form stable covalent adducts with G4 structures via the reactive intermediate, quinone methide (QM). We thoroughly investigated the photochemical properties of the ligands and their ability to generate QMs. Subsequently, we analyzed their specificity for various topologies of G4 and discovered a preferential binding towards the human telomeric sequence. Finally, we tested the ligand ability to act as photochemical alkylating agents, identifying the covalent adducts with G4 structures. This work introduces a novel molecular tool in the chemical biology toolkit for G4s.     

Quadruplex–Duplex Junction: A High-Affinity Binding Site for Indoloquinoline Ligands

A parallel quadruplex derived from the Myc promoter sequence was extended by a stem-loop duplex at either its 5′- or 3′-terminus to mimic a quadruplex–duplex (Q–D) junction as a potential genomic target. High-resolution structures of the hybrids demonstrate continuous stacking of the duplex on the quadruplex core without significant perturbations. An indoloquinoline ligand carrying an aminoalkyl side chain was shown to bind the Q–D hybrids with a very high affinity in the order K_a≈10⁷ m ⁻¹ irrespective of the duplex location at the quadruplex 3′- or 5′-end. NMR chemical shift perturbations identified the tetrad face of the Q–D junction as specific binding site for the ligand. However, calorimetric analyses revealed significant differences in the thermodynamic profiles upon binding to hybrids with either a duplex extension at the quadruplex 3′- or 5′-terminus. A large enthalpic gain and considerable hydrophobic effects are accompanied by the binding of one ligand to the 3′-Q–D junction, whereas non-hydrophobic entropic contributions favor binding with formation of a 2:1 ligand-quadruplex complex in case of the 5′-Q–D hybrid.     

Ligand Design to Acquire Specificity to Intended G-Quadruplex Structures

A G-quadruplex is a nucleic acid secondary structure that is adopted by guanine-rich sequences, and is considered to be relevant in various pharmacological and biological contexts. G-Quadruplexes have also attracted great attention in the field of DNA nanotechnology because of their extremely high thermal stability and the availability of many defined structures. To date, a large repertory of DNA/RNA G-quadruplex-interactive ligands has been developed by numerous laboratories. Several relevant reviews have also been published that have helped researchers to grasp the full scope of G-quadruplex research from its outset to the present. This review focuses on the G-quadruplex ligands that allow targeting of specific G-quadruplexes. Moreover, unique ligands, successful methodologies, and future perspectives in relation to specific G-quadruplex recognition are also addressed.     

Versatile and Homogeneous DNA Tetraplex Platform for Constructing Label-Free Logic Devices: From Design to Application

The design of DNA-based logic circuits has become an active research field in DNA nanotechnology and holds great potential in intelligent bioanalysis. To date, although many DNA-based logic systems have been realized, the implementation of advanced logic functions is still challenging, especially with simple and homogeneous compositions. Herein, by integrating two DNA tetraplex structures (G-quadruplex and i-motif), a completely label-free logic platform with high scalability was established, with which a series of advanced functions were realized, including arithmetic (adders and subtractors) and nonarithmetic ones (majority and dual-transfer gates). Furthermore, the platform was also applied as an intelligent biosensor to coanalyze two cancer-related micro-RNAs with high sensitivities and specificities. Considering the excellent versatility, expandability, and biocompatibility, the platform may promote the development of DNA computing and hold great potential in multiparameter sensing and medical diagnosis.     

One Terminal Guanosine Flip of Intramolecular Parallel G-Quadruplex: Catalytic Enhancement of G-Quadruplex/Hemin DNAzymes

Numerous studies have shown compelling evidence that incorporation of an inversion of polarity site (IPS) in G-rich sequences can affect the topological and structural characteristics of G-quadruplexes (G4s). Herein, the influence of IPS on the formation of a previously studied intramolecular parallel G4 of d(G₃TG₃TG₃TG₃) (TTT) and its stacked higher-order structures is explored. Insertion of 3′–3′ or 5′–5′ IPS did not change the parallel folding pattern of TTT. However, both the species and position of the IPS in TTT have a significant impact on the G4 stability and end-stacking through the alteration of G4–G4 interfaces properties. The data demonstrate that one base flip in each terminal G-tetrad can stabilize parallel G4s and facilitate intermolecular packing of monomeric G4s. Such modifications can also enhance the fluorescence and enzymatic performances by promoting interactions between parallel G4s with N-methyl mesoporphyrin IX (NMM) and hemin, respectively.     

Study of the Function of G‐Rich Aptamers Selected for Lung Adenocarcinoma

Guanine (G)‐rich oligonucleotides have attracted considerable interest as therapeutic agents. Two G‐rich aptamers were selected against epidermal growth factor receptor (EGFR)‐transfected A549 cells, and their G‐rich domains (S13 and S50) were identified to account for the binding of parental aptamers. Circular dichroism (CD) spectra showed that S13 and S50 bound to their targets by forming parallel quadruplexes. Their binding, internalization, and antiproliferation activity in cancer and noncancer cells were investigated by flow cytometry and 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) assay, and compared with those of nucleolin‐binding AS1411 and thrombin‐binding aptamer. The two truncated aptamers (S13 and S50) have good binding and internalization in cancer cells and noncancer cells; however, only S50, similar to AS1411, shows potent antiproliferation against cancer cells. Our data suggest that tumor‐selective antiproliferation of G‐rich oligonucleotides does not directly depend on the binding of the G‐rich aptamer to cells.     

Affinity Chromatography-Based Assays for the Screening of Potential Ligands Selective for G-Quadruplex Structures

DNA G-quadruplexes (G4s) are key structures for the development of targeted anticancer therapies. In this context, ligands selectively interacting with G4s can represent valuable anticancer drugs. Aiming at speeding up the identification of G4-targeting synthetic or natural compounds, we developed an affinity chromatography-based assay, named G-quadruplex on Oligo Affinity Support (G4-OAS), by synthesizing G4-forming sequences on commercially available polystyrene OAS. Then, due to unspecific binding of several hydrophobic ligands on nude OAS, we moved to Controlled Pore Glass (CPG). We thus conceived an ad hoc functionalized, universal support on which both the on-support elongation and deprotection of the G4-forming oligonucleotides can be performed, along with the successive affinity chromatography-based assay, renamed as G-quadruplex on Controlled Pore Glass (G4-CPG) assay. Here we describe these assays and their applications to the screening of several libraries of chemically different putative G4 ligands. Finally, ongoing studies and outlook of our G4-CPG assay are reported.     

An Aggregating Amphiphilic Squaraine: A Light-up Probe That Discriminates Parallel G-Quadruplexes

G-quadruplexes (G4s) are peculiar DNA or RNA tertiary structures that are involved in the regulation of many biological events within mammalian cells, bacteria, and viruses. Although their role as versatile therapeutic targets has been emphasized for 35 years, G4 selectivity over ubiquitous double-stranded DNA/RNA, as well as G4 differentiation by small molecules, still remains challenging. Here, a new amphiphilic dicyanovinyl-substituted squaraine, SQgl , is reported to act as an NIR fluorescent light-up probe discriminating an extensive panel of parallel G4s while it is non-fluorescent in the aggregated state. The squaraine can form an unconventional sandwich π-complex binding two quadruplexes, which leads to a strongly fluorescent (Φ_F=0.61) supramolecular architecture. SQgl is highly selective against non-quadruplex and non-parallel G4 sequences without altering their topology, as desired for applications in selective in vivo high-resolution imaging and theranostics.     

Copper‐Induced Topology Switching and Thrombin Inhibition with Telomeric DNA G‐Quadruplexes

The topological diversity of DNA G‐quadruplexes may play a crucial role in its biological function. Reversible control over a specific folding topology was achieved by the synthesis of a chiral, glycol‐based pyridine ligand and its fourfold incorporation into human telomeric DNA by solid‐phase synthesis. Square‐planar coordination to a Cu^II ion led to the formation of a highly stabilizing intramolecular metal–base tetrad, substituting one G‐tetrad in the parent unimolecular G‐quadruplex. For the Tetrahymena telomeric repeat, Cu^II‐triggered switching from a hybrid‐dominated conformer mixture to an antiparallel topology was observed. Cu^II‐dependent control over a protein–G‐quadruplex interaction was shown for the thrombin–tba pair (tba=thrombin‐binding aptamer).     

Label-free and enzyme-free sensitive fluorescent detection of human immunodeficiency virus deoxyribonucleic acid based on hybridization chain reaction

A label-free and enzyme-free sensitive fluorescent detection of human immunodeficiency virus (HIV) deoxyribonucleic acid (DNA) based on isothermal hybridization chain reaction (HCR) was developed. A G-quadruplex sequence which was incorporated into one of the two hairpin probes was inactive in the absence of target DNA. However, at the presence of target DNA numerous G-quadruplexes along DNA nanowires were self-assembled through HCR. Using N-methyl mesoporphyrin IX (NMM) as the fluorophore, a “turn-on” fluorescent response would be achieved and detected as low as 0.5 nmol L⁻¹ of HIV DNA. This proposed method was applied to detect HIV DNA in biologic samples with satisfactory results.