When annealing is detrimental: The case of HMGB1-targeting G-quadruplex aptamers.

In this work, we present the case of the G-quadruplex(G4)-forming aptamers we recently identified for the recognition of HMGB1, protein involved in inflammation, autoimmune diseases and cancer. These aptamers were previously analyzed, without annealing them, after proper dilution of the stock solution in a pseudo-physiological buffer mimicking the extracellular environment where the protein exerts its pathological activity, and showed high thermal stability and nuclease resistance, good protein affinity and remarkable in vitro activity. These features were more marked for the aptamers forming dimeric, parallel G4 structures in solution.

Experimental and Computational Evidence of a Stable RNA G-Triplex Structure at Physiological Temperature in the SARS-CoV-2 Genome.

RG1 is a quadruplex-forming sequence in the SARS-CoV-2 genome proposed as possible therapeutic target for COVID-19. We demonstrate that the dominant conformation of RG1 under physiological conditions differs from the parallel quadruplex previously assumed. Through comprehensive investigations employing CD, UV, NMR, DSC, gel electrophoresis, MD simulations, in silico spectroscopy and the use of truncated RG1 sequences, we have identified this stable conformation as an RNA G-triplex composed of two G-triads.

Probing naphthalene diimide and 3-hydroxypropylphosphate as end-conjugating moieties for improved thrombin binding aptamers: Structural and biological effects.

The limitations associated with the in vivo use of the thrombin binding aptamer (TBA or TBA) have dramatically stimulated the search of suitable chemically modified analogues in order to discover effective and reversible inhibitors of thrombin activity. In this context, we previously proposed cyclic and pseudo-cyclic TBA analogues with improved stability that proved to be more active than the parent aptamer. Herein, we have investigated a novel library of TBA derivatives carrying naphthalene diimide (NDI) moieties at the 3'- or 5'-end.

Design, Synthesis, and Characterization of an Amphiphilic Lipoic Acid-Based Ru(III) Complex as a Versatile Tool for the Functionalization of Different Nanosystems.

Ru-based chemotherapy is emerging as an effective alternative to the well-established Pt-based one, typically associated with high toxicity. In this context, our recent efforts were devoted to the preparation of nucleolipid-based Ru(III) complexes able to form, under physiological conditions, supramolecular aggregates which can efficiently prevent metal deactivation and convey Ru(III) inside the cells where it exerts its activity. Within an interdisciplinary program for the development of multifunctional nanoparticles for theranostic applications, we here report the design, synthesis, and characterization of a novel functionalized Ru(III) salt, carrying a lipoic acid moiety in the nucleolipid-based scaffold to allow its incorporation onto metal-based nanoparticles.

Exploring the Binding of Natural Compounds to Cancer-Related G-Quadruplex Structures: From 9,10-Dihydrophenanthrenes to Their Dimeric and Glucoside Derivatives.

In-depth studies on the interaction of natural compounds with cancer-related G-quadruplex structures have been undertaken only recently, despite their high potential as anticancer agents, especially due to their well-known and various bioactivities. In this frame, aiming at expanding the repertoire of natural compounds able to selectively recognize G-quadruplexes, and particularly focusing on phenanthrenoids, a mini-library including dimeric (-) and glucoside (-) analogues of 9,10-dihydrophenanthrenes, a related tetrahydropyrene glucoside () along with 9,10-dihydrophenanthrene were investigated here by several biophysical techniques and molecular docking. Compounds and emerged as the most selective G-quadruplex ligands within the investigated series.

Selective Targeting of Cancer-Related G-Quadruplex Structures by the Natural Compound Dicentrine.

Aiming to identify highly effective and selective G-quadruplex ligands as anticancer candidates, five natural compounds were investigated here, i.e., the alkaloids Canadine, D-Glaucine and Dicentrine, as well as the flavonoids Deguelin and Millettone, selected as analogs of compounds previously identified as promising G-quadruplex-targeting ligands.

Insights into the Small Molecule Targeting of Biologically Relevant G-Quadruplexes: An Overview of NMR and Crystal Structures.

G-quadruplexes turned out to be important targets for the development of novel targeted anticancer/antiviral therapies. More than 3000 G-quadruplex small-molecule ligands have been described, with most of them exerting anticancer/antiviral activity by inducing telomeric damage and/or altering oncogene or viral gene expression in cancer cells and viruses, respectively. For some ligands, in-depth NMR and/or crystallographic studies were performed, providing detailed knowledge on their interactions with diverse G-quadruplex targets.

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.

Natural compounds from plants interacting with telomeric and oncogene G-quadruplex structures as potential anticancer agents.

Aiming at discovering novel, putative anticancer drugs featuring low-to-null side effects, natural compounds isolated from were studied here for their ability to target G-quadruplex structures originating from cancer-related telomeric and oncogene DNA sequences. Particularly, various dihydrophenanthrene, benzocoumarin and dihydrodibenzoxepin derivatives were firstly screened by the affinity chromatography-based G4-CPG assay, and the compound with the highest affinity and selectivity for G-quadruplexes (named J10) was selected for further studies. Fluorescence spectroscopy and circular dichroism experiments corroborated its capability to selectively recognize and stabilize G-quadruplexes over duplex DNA, also showing a preference for parallel G-quadruplexes.

Identification of Effective Anticancer G-Quadruplex-Targeting Chemotypes through the Exploration of a High Diversity Library of Natural Compounds.

In the quest for selective G-quadruplex (G4)-targeting chemotypes, natural compounds have been thus far poorly explored, though representing appealing candidates due to the high structural diversity of their scaffolds. In this regard, a unique high diversity in-house library composed of ca. one thousand individual natural products was investigated.

DNA Binding Mode Analysis of a Core-Extended Naphthalene Diimide as a Conformation-Sensitive Fluorescent Probe of G-Quadruplex Structures.

G-quadruplex existence was proved in cells by using both antibodies and small molecule fluorescent probes. However, the G-quadruplex probes designed thus far are structure- but not conformation-specific. Recently, a core-extended naphthalene diimide () was designed and found to provide fluorescent signals of markedly different intensities when bound to G-quadruplexes of different conformations or duplexes.

Interference of Polydatin/Resveratrol in the ACE2:Spike Recognition during COVID-19 Infection. A Focus on Their Potential Mechanism of Action through Computational and Biochemical Assays.

In the search for new therapeutic strategies to contrast SARS-CoV-2, we here studied the interaction of polydatin (PD) and resveratrol (RESV)-two natural stilbene polyphenols with manifold, well known biological activities-with Spike, the viral protein essential for virus entry into host cells, and ACE2, the angiotensin-converting enzyme present on the surface of multiple cell types (including respiratory epithelial cells) which is the main host receptor for Spike binding. Molecular Docking simulations evidenced that both compounds can bind Spike, ACE2 and the ACE2:Spike complex with good affinity, although the interaction of PD appears stronger than that of RESV on all the investigated targets. Preliminary biochemical assays revealed a significant inhibitory activity of the ACE2:Spike recognition with a dose-response effect only in the case of PD.

From Prebiotic Chemistry to Supramolecular Biomedical Materials: Exploring the Properties of Self-Assembling Nucleobase-Containing Peptides.

Peptides and their synthetic analogs are a class of molecules with enormous relevance as therapeutics for their ability to interact with biomacromolecules like nucleic acids and proteins, potentially interfering with biological pathways often involved in the onset and progression of pathologies of high social impact. Nucleobase-bearing peptides (nucleopeptides) and pseudopeptides (PNAs) offer further interesting possibilities related to their nucleobase-decorated nature for diagnostic and therapeutic applications, thanks to their reported ability to target complementary DNA and RNA strands. In addition, these chimeric compounds are endowed with intriguing self-assembling properties, which are at the heart of their investigation as self-replicating materials in prebiotic chemistry, as well as their application as constituents of innovative drug delivery systems and, more generally, as novel nanomaterials to be employed in biomedicine.

Plant-Derived Stilbenoids as DNA-Binding Agents: From Monomers to Dimers.

Stilbenoids are natural compounds endowed with several biological activities, including cardioprotection and cancer prevention. Among them, (±)-trans-δ-viniferin, deriving from trans-resveratrol dimerization, was investigated in its ability to target DNA duplex and G-quadruplex structures by exploiting NMR spectroscopy, circular dichroism, fluorescence spectroscopy and molecular docking. (±)-trans-δ-Viniferin proved to bind both the minor and major grooves of duplexes, whereas it bound the 3'- and 5'-ends of a G-quadruplex by stacking on the outer quartets, accompanied by rearrangement of flanking residues.

Disentangling the Structure-Activity Relationships of Naphthalene Diimides as Anticancer G-Quadruplex-Targeting Drugs.

In the context of developing efficient anticancer therapies aimed at eradicating any sort of tumors, G-quadruplexes represent excellent targets. Small molecules able to interact with G-quadruplexes can interfere with cell pathways specific of tumors and common to all cancers. Naphthalene diimides (NDIs) are among the most promising, putative anticancer G-quadruplex-targeting drugs, due to their ability to simultaneously target multiple G-quadruplexes and their strong, selective in vitro and in vivo anticancer activity.

On the interaction of an anticancer trisubstituted naphthalene diimide with G-quadruplexes of different topologies: a structural insight.

Naphthalene diimides showed significant anticancer activity in animal models, with therapeutic potential related to their ability to strongly interact with G-quadruplexes. Recently, a trifunctionalized naphthalene diimide, named NDI-5, was identified as the best analogue of a mini-library of novel naphthalene diimides for its high G-quadruplex binding affinity along with marked, selective anticancer activity, emerging as promising candidate drug for in vivo studies. Here we used NMR, dynamic light scattering, circular dichroism and fluorescence analyses to investigate the interactions of NDI-5 with G-quadruplexes featuring either parallel or hybrid topology.

On the binding of naphthalene diimides to a human telomeric G-quadruplex multimer model.

To selectively target telomeric G-quadruplex (G4) DNA, monomeric and dimeric naphthalene diimides (NDIs) were investigated as binders of multimeric G4 structures able to discriminate duplex DNA. These NDIs were analysed by the affinity chromatography-based screening G4-CPG (G-quadruplex on Controlled Pore Glass), using the sequence d[AGGG(TTAGGG)] (tel46), folding into two consecutive G4s, as model of the human telomeric G4 multimer. In parallel, a telomeric G4 monomer (tel26) and a duplex structure (ds27) were used as controls.

Anti-VEGF DNA-based aptamers in cancer therapeutics and diagnostics.

The vascular endothelial growth factor (VEGF) family and its receptors play fundamental roles not only in physiological but also in pathological angiogenesis, characteristic of cancer progression. Aiming at finding putative treatments for several malignancies, various small molecules, antibodies, or protein-based drugs have been evaluated in vitro and in vivo as VEGF inhibitors, providing efficient agents approved for clinical use. Due to the high clinical importance of VEGF, also a great number of anti-VEGF nucleic acid-based aptamers-that is, oligonucleotides able to bind with high affinity and specificity a selected biological target-have been developed as promising agents in anticancer strategies.

Synthesis, Antiproliferative Activity, and DNA Binding Studies of Nucleoamino Acid-Containing Pt(II) Complexes.

We here report our studies on the reaction with the platinum(II) ion of a nucleoamino acid constituted by the l-2,3-diaminopropanoic acid linked to the thymine nucleobase through a methylenecarbonyl linker. The obtained new platinum complexes, characterized by spectroscopic and mass spectrometric techniques, were envisaged to exploit synergistic effects due to the presence of both the platinum center and the nucleoamino acid moiety. The latter can be potentially useful to protect the complexes from early deactivation, as well as to facilitate their cell internalization.

G-quadruplex-based aptamers targeting human thrombin: Discovery, chemical modifications and antithrombotic effects.

First studies on thrombin-inhibiting DNA aptamers were reported in 1992, and since then a large number of anticoagulant aptamers has been discovered. TBA - also named HD1, a 15-mer G-quadruplex (G4)-forming oligonucleotide - is the best characterized thrombin binding aptamer, able to specifically recognize the protein exosite I, thus inhibiting the conversion of soluble fibrinogen into insoluble fibrin strands. Unmodified nucleic acid-based aptamers, in general, and TBA in particular, exhibit limited pharmacokinetic properties and are rapidly degraded in vivo by nucleases.

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs.

In the optimization process of nucleic acid aptamers, increased affinity and/or activity are generally searched by exploring structural analogues of the lead compound. In many cases, promising results have been obtained by dimerization of the starting aptamer. Here we studied a focused set of covalent dimers of the G-quadruplex (G4) forming anti-Vascular Endothelial Growth Factor (VEGF) V7t1 aptamer with the aim of identifying derivatives with improved properties.

Trifunctionalized Naphthalene Diimides and Dimeric Analogues as G-Quadruplex-Targeting Anticancer Agents Selected by Affinity Chromatography.

A focused library of newly designed monomeric and dimeric naphthalene diimides (NDIs) was analyzed in its ability to recognize specific G-quadruplex (G4) structures discriminating duplex DNA. The best G4 ligands-according to an affinity chromatography-based screening method named G4-CPG-were tested on human cancer and healthy cells, inducing DNA damage at telomeres, and in parallel, showing selective antiproliferative activity on HeLa cancer cells with IC values in the low nanomolar range. CD and fluorescence spectroscopy studies allowed detailed investigation of the interaction in solution with different G4 and duplex DNA models of the most promising NDI of the series, as determined by combining the biophysical and biological assays' data.

Synthesis, DNA binding studies, and antiproliferative activity of novel Pt(II)-complexes with an L-alanyl-based ligand.

An artificial alanine-based amino acid {(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoic acid, here named TioxAla}, bearing a substituted triazolyl-thione group on the side chain and able to bind RNA biomedical targets, was here chosen as a valuable scaffold for the synthesis of new platinum complexes with potential dual action owing to the concomitant presence of the metal centre and the amino acid moiety. Three new platinum complexes, obtained from the reaction of TioxAla with KPtCl, were characterized by mass spectrometry, nuclear magnetic resonance and UV-vis spectroscopy: one compound (Pt1, bis-{(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoate-O,S} platinum(II)) consisted of two amino acid units coordinating the Pt(II) ion; the other two, Pt2 [potassium dichloro-{(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoate (O,S)} platinum(II)] and Pt3 [potassium dichloro-{(S)-2-amino-3-[4-propyl-3-(thiophen-2-yl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]propanoate (O,N)} platinum(II)], were isomers bearing one TioxAla unit, and two chlorides as Pt-ligands. Pt coordination involved preferentially the amino, carboxylic and thione functions of TioxAla.

Shedding light on the interaction of polydatin and resveratrol with G-quadruplex and duplex DNA: a biophysical, computational and biological approach.

Among polyphenols, trans-resveratrol (tRES) and trans-polydatin (tPD) exert multiple biological effects, particularly antioxidant and antiproliferative. In this work, we have investigated the interaction of tPD with three cancer-related DNA sequences able to form G-quadruplex (G4) structures, as well as with a model duplex, and compared its behaviour with tRES. Interestingly, fluorescence analysis evidenced the ability of tPD to bind all the studied DNA systems, similarly to tRES, with tRES displaying a higher ability to discriminate G4 over duplex with respect to tPD.

The role of G-quadruplex structures of LIGS-generated aptamers R1.2 and R1.3 in IgM specific recognition.

Exploiting a variant of SELEX called "Ligand-Guided Selection" (LI-GS), we recently identified two novel truncated G-rich aptamers, called R1.2 and R1.3, specific for membrane-bound IgM (mIgM), the hallmark of B cells.