Nucleic acid aptamers are single-stranded DNA or RNA molecules identified to recognize with high affinity specific targets including proteins, small molecules, ions, whole cells and even entire organisms, such as viruses or bacteria. They can be identified from combinatorial libraries of DNA or RNA oligonucleotides by SELEX technology, an in vitro iterative selection procedure consisting of binding (capture), partitioning and amplification steps. Remarkably, many of the aptamers selected against biologically relevant protein targets are G-rich sequences that can fold into stable G-quadruplex (G4) structures. Aiming at disseminating novel inspiring ideas within the scientific community in the field of G4-structures, the emphasis of this review is placed on: 1) recent advancements in SELEX technology for the efficient and rapid identification of new candidate aptamers (introduction of microfluidic systems and next generation sequencing); 2) recurrence of G4 structures in aptamers selected by SELEX against biologically relevant protein targets; 3) discovery of several G4-forming motifs in important regulatory regions of the human or viral genome bound by endogenous proteins, which per se can result into potential aptamers; 4) an updated overview of G4-based aptamers with therapeutic potential and 5) a discussion on the most attractive G4-based aptamers for diagnostic applications. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Looking for new metal-based anticancer treatments, in recent years many ruthenium complexes have been proposed as effective and safe potential drugs. In this context we have recently developed a novel approach for the in vivo delivery of Ru(III) complexes, preparing stable ruthenium-based nucleolipidic nanoaggregates endowed with significant antiproliferative activity. Herein we describe the cellular response to our ruthenium-containing formulations in selected models of human breast cancer. By in vitro bioscreens in the context of preclinical studies, we have focused on their ability to inhibit breast cancer cell proliferation by the activation of the intrinsic apoptotic pathway, possibly via mitochondrial perturbations involving Bcl-2 family members and predisposing to programmed cell death. In addition, the most efficient ruthenium-containing cationic nanoaggregates we have hitherto developed are able to elicit both extrinsic and intrinsic apoptosis, as well as autophagy. To limit chemoresistance and counteract uncontrolled proliferation, multiple cell death pathways activation by metal-based chemotherapeutics is a challenging, yet very promising strategy for targeted therapy development in aggressive cancer diseases, such as triple-negative breast cancer with limited treatment options. These outcomes provide valuable, original knowledge on ruthenium-based candidate drugs and new insights for future optimized cancer treatment protocols.

According to WHO, breast cancer incidence is increasing so that the search for novel chemotherapeutic options is nowadays an essential requirement to fight neoplasm subtypes. By exploring new effective metal-based chemotherapeutic strategies, many ruthenium complexes have been recently proposed as antitumour drugs, showing ability to impact on diverse cellular targets. In the framework of different molecular pathways leading to cell death in human models of breast cancer, here we demonstrate autophagy involvement behind the antiproliferative action of a ruthenium(III)-complex incorporated into a cationic nanosystem (HoThyRu/DOTAP), proved to be hitherto one of the most effective within the suite of nucleolipidic formulations we have developed for the in vivo transport of anticancer ruthenium(III)-based drugs. Indeed, evidences are implicating autophagy in both cancer development and therapy, and anticancer interventions endowed with the ability to trigger this biological response are currently considered attractive oncotherapeutic approaches. Moreover, crosstalk between apoptosis and autophagy, regulated by finely tuned metallo-chemotherapeutics, may provide novel opportunities for future improvement of cancer treatment. Following this line, our in vitro and in vivo preclinical investigations suggest that an original strategy based on suitable formulations of ruthenium(III)-complexes, inducing sustained cell death, could open new opportunities for breast cancer treatment, including the highly aggressive triple-negative subtype.

With the aim of developing an ew approacht oo btain improveda ptamers, ac yclic thrombin-binding aptamer (TBA) analogue (cycTBA) has been prepared by exploiting ac opper(I)assisted azide-alkyne cycloaddition. The markedlyi ncreased serum resistance and exceptional thermal stabilityo ft he Gquadruplexv ersus TBA were associated with halved thrombin inhibition, which suggested that some flexibility in the TBA structurew as necessary for protein recognition.In the panorama of anticoagulant agents, inhibitors of thrombin, which is a" trypsin-like" serine protease with fundamental roles in blood clottingt oc onvert soluble fibrinogen into insoluble fibrin, [1] are amongt he most reliable andw idely exploitedd rugs against thrombosis. The 15-mer, G-rich, oligonucleotide thrombin-binding aptamer (TBA 15 or simply TBA), which contains the sequence 5'-d(GGTTGGTGTGGTTGG)-3',i s the bestcharacterisedaptamer of thrombin. TBA has been proposed as av aluablea lternative to classical thrombini nhibitors used in the clinic, such as heparin, warfarin, and bivalirudin, which have severe side effects or suffer from narrow therapeutic windows. [2] Upon folding into an antiparallel, chair-like Gquadruplex( G4) structure, TBA can tightly and selectively bind the fibrinogen-binding exosite Io fh uman thrombin, and thus, inhibit its key functions in the coagulation cascade. [3] Due to suboptimal dosing profiles, TBA did not progress to advanced clinicalt rials, but was blockeda fter phase Is tudies. [4] Since then, al arge number of TBA analogues have been synthesised with either backbonem odifications [5] or integrated into different nanosystems, includingm agnetic, [6] gold [7] and silica nanoparticles. [8] Although many of thesea nalogues have shown promising pharmacokinetic profiles, none have thus far reachedi nv ivo studies.As ag eneral strategy to improve the in vivo properties of TBA, we herein propose ac yclisation approach to obtain novel, better performing TBA analogues.T his approach involves the covalent connection, through ap roper flexible linker,o ft he 3'-a nd 5'-ends of the oligonucleotide strand.T wo major benefits are expectedu pon TBA cyclisation:o no ne hand, the absence of the 3' and 5' terminis hould sensibly protect the oligonucleotide from nuclease degradation; thus significantly prolongingi ts in vivo half-life;ont he otherhand, the cyclic backbone should imposeastructural preorganisation of the aptamer andf avour G4 formation, stabilising this conformation, which is the effectively bioactive one, thus enhancing its target affinity. This approach has been extensivelya dopted in the past to improvet he general properties of peptides [9] and peptidomimetics, [10] as well as peptide nucleic acids (PNAs) [11] and glycomimetics, [12] but has only been applied in al imited extent to oligonucleotides, [13] in general, and, to the best of our knowledge, is essentially unexploited thus far on aptamers.Herein, we report the design, synthesis and biophysical characterisation of an unprecedentedc yclic TBA analogue,...

Ruthenium complexes are attracting increasing attention as second‐generation metal‐based anticancer agents, with NAMI‐A and KP1019 as the major representatives of this class having undergone clinical trials. Our recent interest has been focused on the synthesis and characterization of new amphiphilic derivatives of nucleosides (nucleolipids). These compounds have been selected as core scaffolds linked to RuIII complexes and capable of self‐assembly into stable nanostructures in aq. solutions so to transport the metal ions efficiently into the cell. Through the use of ribo‐ and deoxyribonucleosides as starting building blocks, a mini‐library of nucleolipidic RuIII complexes decorated with diverse hydrophilic and lipophilic chains and incorporating the NAMI‐A analogue AziRu has been prepared. When co‐aggregated with biocompatible lipids, these RuIII‐containing nucleolipids proved to be stable for months under physiological conditions. Detailed microstructural characterization, carried out by a combined approach including different physico‐chemical techniques, allowed their stability, size and shape to be determined. Tested on a panel of human and non‐human cells, all of the studied RuIII complexes showed potent in vitro anticancer activity, significantly higher than that of NAMI‐A‐like analogues, and minimal toxicity. Here we summarize the design and synthetic procedures developed to prepare these new Ru‐containing candidate drugs, discussing the beneficial effects achievable through exploiting nucleolipid appendages in the delivery of metal‐based drugs.