Design, Synthesis and Characterization of Cyclic NU172 Analogues: A Biophysical and Biological Insight

Abstract

NU172—a 26-mer oligonucleotide able to bind exosite I of human thrombin and inhibit its activity—was the first aptamer to reach Phase II clinical studies as an anticoagulant in heart disease treatments. With the aim of favoring its functional duplex-quadruplex conformation and thus improving its enzymatic stability, as well as its thrombin inhibitory activity, herein a focused set of cyclic NU172 analogues—obtained by connecting its 5′- and 3′-extremities with flexible linkers—was synthesized. Two different chemical approaches were exploited in the cyclization procedure, one based on the oxime ligation method and the other on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC), affording NU172 analogues including circularizing linkers with different length and chemical nature. The resulting cyclic NU172 derivatives were characterized using several biophysical techniques (ultraviolet (UV) and circular dichroism (CD) spectroscopies, gel electrophoresis) and then investigated for their serum resistance and anticoagulant activity in vitro. All the cyclic NU172 analogues showed higher thermal stability and nuclease resistance compared to unmodified NU172. These favorable properties were, however, associated with reduced—even though still significant—anticoagulant activity, suggesting that the conformational constraints introduced upon cyclization were somehow detrimental for protein recognition. These results provide useful information for the design of improved analogues of NU172 and related duplex-quadruplex structures.

Charge-Transfer Interactions Stabilize G-Quadruplex-Forming Thrombin Binding Aptamers and Can Improve Their Anticoagulant Activity

“…This approach was then extended to NU172, another anticoagulant aptamer [48,49], with which we varied the length and chemical properties of the linker connecting the extremities of the G4 domain, thereby obtaining a focused set of cyclic NU172 derivatives. However, in all cases, a lower inhibitory activity than that of the unmodified aptamer was obtained [50]. These outcomes suggested that increased rigidity of the G4 structure-both in TBA and NU172-as a consequence of the cyclic structure, could somehow reduce the aptamer's affinity for the protein, and thus its bioactivity.…”

“…The end-functionalized TBAs were tested for their anticoagulant activity with respect to unmodified TBA by means of light scattering (LS) experiments, by following the thrombin-catalyzed conversion of fibrinogen into fibrin in PBS, used to mimic physiological conditions [46,47,49,50,93]. Normalized LS intensity curves, as a function of time, allow following the fibrinogen-fibrin conversion because the intensity of the scattered light depends on the size of the objects in the solution.…”

“…The CD melting curves showed sigmoidal profiles and were used to determine the thermodynamic change values using the van't Hoff analysis, as previously performed [50]. In detail, ∆H 0 and ∆S 0 values were calculated from the experimental CD profiles by the Marquardt nonlinear least-squares method used by Petersheim and Turner [97] adapted to a monomolecular system [79], under the assumption ∆Cp = 0.…”

“…Denaturing PAGE. Denaturing PAGE experiments were performed according to reported procedures [50,98], with minor modifications. Briefly, 105 pmol of each oligonucleotide sample in water were mixed with formamide (1:2, v/v) to completely unfold the samples, heated at 95 • C for 5 min, then left in contact with ice until subsequent loading.…”

Dimeric and Multimeric DNA Aptamers for Highly Effective Protein Recognition

“…In order to simultaneously increase the resistance to nuclease degradation and the thrombin binding properties, Di Giusto and colleagues proposed circular multivalent constructs [ 117 , 118 ]. Indeed, circularization is a well-established strategy to improve the performance of aptamers [ 119 , 120 ] and has been efficiently applied to antithrombin aptamers targeting thrombin exosite I [ 96 , 121 , 122 , 123 ].…”