Stability Is Not Everything: The Case of the Cyclisation of a Thrombin‐Binding Aptamer

Abstract

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,...

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

“…Indeed, gel mobility of G4 structures is essentially affected by their conformation and compactness provided that their mass/charge ratio is quite similar. Since this is our case, PAGE results suggested a more compact structure for all the covalent V7t1 dimers, producing a faster migration on the gel [43,44].…”

Structure–Activity Relationship Study of a Potent α‐Thrombin Binding Aptamer Incorporating Hexitol Nucleotides

“…[9] Among the many strategies devised to overcome this long-standing limitation, [10,12] the chemical modification of the aptamer structure has been one of the most studied approaches, [28,29] involving changes in the sugar backbone, [30][31][32][33][34][35][36][37] the internucleotide linkages, [38][39][40] or sequence/length variations of key residues. [41][42][43][44] However, while modifications have been mainly focused on HD1, [30][31][32][33][34][36][37][38][39][40][41][42][43][44] no changes in the structure of NU172 have been reported so far. In this context, in the frame of a research program aimed to explore the biomimetic potential of various synthetic pairing systems, [45][46][47][48][49] we have focused attention on the analysis of the structural and functional changes in NU172 following the incorporation of unnatural nucleotides belonging to the class of Hexitol Nucleic Acids (HNA).…”

Fine-tuning the properties of the thrombin binding aptamer through cyclization: Effect of the 5′-3′ connecting linker on the aptamer stability and anticoagulant activity

“…[41] As a proof-of-concept, we have previously synthesized a cyclic TBA analogue obtained by covalently connecting the 3′-and 5′-ends of this G-rich oligonucleotide with a 20 atom long linker, ensuring approximately the same length of a trinucleotide (Figure 1). [42] cyclic TBA TBA vs. The biophysical characterization of this first cyclic TBA prototype, that we named cycTBA, highlighted the noteworthy advantages obtained upon cyclization of the aptamer: indeed, a remarkable increase in the thermal stability of its G-quadruplex structure (ΔT m of ca.…”

“…180-fold higher half-life in PBS) were observed, denoting a much more compact and tightly assembled structure compared to unmodified TBA. [42] This cyclic TBA analogue was obtained by covalently linking the 3′-and 5′-ends of the TBA sequence (Figure 1), which in the folded antiparallel G4 structure point in the same direction and are far away from the TT loops recognized by the protein. [43][44][45] Thus, the presence of a circularizing linker in principle should not impair the recognition and interaction of the aptamer with the target protein.…”