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,...

Biomolecular condensates formed by liquid–liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming nonmembranous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the subseafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.

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Cationic antimicrobial peptides (CAMPs) are a promising alternative to treat multidrug-resistant bacteria, which have developed resistance to all the commonly used antimicrobial, and therefore represent a serious threat to human health. One of the major drawbacks of CAMPs is their sensitivity to proteases, which drastically limits their half-life. Here we describe the design and synthesis of three nine-residue CAMPs, which showed high stability in serum and broad spectrum antimicrobial activity. As for all peptides a very low selectivity between bacterial and eukaryotic cells was observed, we performed a detailed biophysical characterization of the interaction of one of these peptides with liposomes mimicking bacterial and eukaryotic membranes. Our results show a surface binding on the DPPC/DPPG vesicles, coupled with lipid domain formation, and, above a threshold concentration, a deep insertion into the bilayer hydrophobic core. On the contrary, mainly surface binding of the peptide on the DPPC bilayer was observed. These observed differences in the peptide interaction with the two model membranes suggest a divergence in the mechanisms responsible for the antimicrobial activity and for the observed high toxicity toward mammalian cell lines. These results could represent an important contribution to unravel some open and unresolved issues in the development of synthetic CAMPs.

The G-quadruplex-forming VEGF-binding aptamer V7t1 was previously found to be highly polymorphic in a K+-containing solution and, to restrict its conformational preferences to a unique, well-defined form, modified nucleotides (LNA and/or UNA) were inserted in its sequence. We here report an in-depth biophysical characterization of V7t1 in a Na+-rich medium, mimicking the extracellular environment in which VEGF targeting should occur, carried out combining several techniques to analyse the conformational behaviour of the aptamer and its binding to the protein. Our results demonstrate that, in the presence of high Na+ concentrations, V7t1 behaves in a very different way if subjected or not to annealing procedures, as evidenced by native gel electrophoresis, size exclusion chromatography and dynamic light scattering analysis. Indeed, not-annealed V7t1 forms both monomeric and dimeric G-quadruplexes, while the annealed oligonucleotide is a monomeric species. Remarkably, only the dimeric aptamer efficiently binds VEGF, showing higher affinity for the protein compared to the monomeric species. These findings provide new precious information for the development of improved V7t1 analogues, allowing more efficient binding to the cancer-related protein and the design of effective biosensors or theranostic devices based on VEGF targeting.