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.
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.
We investigated the volumetric and kinetic profile of the conformational landscape of a poly dA loop DNA hairpin (Hp) in the presence of salts, osmolytes and crowding media, mimicking the intracellular milieu, using single-molecule FRET methodology. Pressure modulation was applied to explore the volumetric and hydrational characteristics of the free-energy landscape of the DNA Hp, but also because pressure is a stress factor many organisms have to cope with, e.g. in the deep sea where pressures even up to the kbar level are encountered. Urea and pressure synergistically destabilize the closed conformation of the DNA Hp due to a lower molar partial volume in the unfolded state. Conversely, multivalent salts, trimethylamine-N-oxide and Ficoll strongly populate the closed state and counteract deteriorating effects of pressure. Complementary smFRET measurements under immobilized conditions at ambient pressure allowed us to dissect the equilibrium data in terms of folding and unfolding rate constants of the conformational transitions, leading to a deeper understanding of the stabilization mechanisms of the cosolutes. Our results show that the free-energy landscape of the DNA Hp is a rugged one, which is markedly affected by the ionic strength of the solution, by preferential interaction and exclusion of cosolvents as well as by pressure.
Interactions between proteins and ligands, which are fundamental to many biochemical processes essential to life, are mostly studied at dilute buffer conditions. The effects of the highly crowded nature of biological cells and the effects of liquid-liquid phase separation inducing biomolecular droplet formation as a means of membrane-less compartmentalization have been largely neglected in protein binding studies. We investigated the binding of a small ligand (ANS) to one of the most multifunctional proteins, bovine serum albumin (BSA) in an aqueous two-phase system (ATPS) composed of PEG and Dextran. Also, aiming to shed more light on differences in binding mode compared to the neat buffer data, we examined the effect of high hydrostatic pressure (HHP) on the binding process. We observe a marked effect of the ATPS on the binding characteristics of BSA. Not only the binding constants change in the ATPS system, but also the integrity of binding sites is partially lost, which is most likely due to soft enthalpic interactions of the BSA with components in the dense droplet phase of the ATPS. Using pressure modulation, differences in binding sites could be unravelled by their different volumetric and hydration properties. Regarding the vital biological relevance of the study, we notice that extreme biological environments, such as HHP, can markedly affect the binding characteristics of proteins. Hence, organisms experiencing high-pressure stress in the deep sea need to finely adjust the volume changes of their biochemical reactions in cellulo. One of the most common experiments in biochemistry, biophysics, medicinal chemistry, and cellular biology is testing whether a ligand binds to a protein 1-5. Protein-ligand recognition and interaction are fundamental to many events essential to life, such as self-replication, metabolism and signal transduction. Furthermore, elucidating the nature of the forces involved in the binding processes is prerequisite for the development of new and more effective drugs in medical applications. In spite of its apparent importance, many aspects of ligand binding have not been fully explored, yet. Commonly, binding studies are carried out in dilute buffer solution and at ambient temperature and pressure. But the interior of biological cells is enriched with numerous macromolecules, such as proteins and nucleic acids, forming a highly crowded environment. Crowding affects molecular diffusion, conformation, dynamics and kinetics as well as the hydration properties of proteins 6-9. Further, biological cells need to orchestrate their biochemical reactions in space and time. The modulation and regulation of such processes is achieved through the compartmentalization of the cellular milieu. Besides lipid bilayer membranes, non-membrane bound compartments lacking a surrounding lipid bilayer and consisting of phase-separated liquid-like droplets have been shown to be of similar importance in recent years 10,11. Such membrane-less droplet-like compartments, also denoted biomolecular condensates, ar...
Elucidating the details of the formation, stability, interactions, and reactivity of biomolecular systems under extreme environmental conditions, including high salt concentrations in brines and high osmotic and high hydrostatic pressures, is of fundamental biological, astrobiological, and biotechnological importance. Bacteria and archaea are able to survive in the deep ocean or subsurface of Earth, where pressures of up to 1 kbar are reached. The deep subsurface of Mars may host high concentrations of ions in brines, such as perchlorates, but we know little about how these conditions and the resulting osmotic stress conditions would affect the habitability of such environments for cellular life. We discuss the combined effects of osmotic (salts, organic cosolvents) and hydrostatic pressures on the structure, stability, and reactivity of biomolecular systems, including membranes, proteins, and nucleic acids. To this end, a variety of biophysical techniques have been applied, including calorimetry, UV/vis, FTIR and fluorescence spectroscopy, and neutron and X-ray scattering, in conjunction with high pressure techniques. Knowledge of these effects is essential to our understanding of life exposed to such harsh conditions, and of the physical limits of life in general. Finally, we discuss strategies that not only help us understand the adaptive mechanisms of organisms that thrive in such harsh geological settings but could also have important ramifications in biotechnological and pharmaceutical applications.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.