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.

Keywords: DNA aptamers; G-quadruplex; NU172; anticoagulant activity; biophysical characterization; cyclization; duplex/quadruplex; structure-activity relationship; thrombin.

Figure 1

Schematic representation of the antiparallel G-quadruplex structure of TBA (a), the duplex/quadruplex structure of NU172 (b), its minimal G4 motif indicated as NU (c) and a general scheme for cyclic NU172 analogues (d) with a linker connecting the 3′- and 5′-ends of the oligonucleotide sequence. The guanines involved in the G-quadruplex core formation are highlighted in light blue, while the duplex module is depicted in green. In both NU and NU172 structures, the nucleobases of the G4 core differing from TBA are indicated in dark red.

Figure 2

Representative normalized TDS (a,b) and UV-melting profiles (c,d) of the cyclic NU172 derivatives (light blue, orange, green, and magenta lines, respectively for cycNU172-EG2, cycNU172-EG3, cycNU172-Ph and cycNU172-Pro). All the oligonucleotide samples were analyzed at 2 µM concentration in both the selected K⁺- (a,c) and Na⁺-rich (b,d) buffer solutions, in comparison with unmodified NU172 and NU (dark red and black lines, respectively). TDS profiles result from the subtraction of the UV spectrum registered at 15 °C from the 90 °C one. UV-melting profiles—recorded at 295 nm using a scan rate of 1 °C/min—are reported as normalized absorbance as a function of the temperature.

Figure 3

Representative overlapped CD spectra (a,b) and CD-melting profiles (c,d) of the cyclic NU172 derivatives (light blue, orange, green, and magenta lines, respectively for cycNU172-EG2, cycNU172-EG3, cycNU172-Ph and cycNU172-Pro), recorded at 2 µM concentration and 15 °C in both the selected K⁺- (a,c) and Na⁺-rich (b,d) buffer solutions, in comparison with unmodified NU172 and NU (dark red and black lines, respectively). CD-melting profiles—recorded at the maximum ellipticity observed for each oligonucleotide system, using a scan rate of 1 °C/min—are reported as folded fraction of each oligonucleotide system as a function of temperature.

Figure 4

Representative 15% polyacrylamide gel electrophoresis under native conditions of the oligonucleotide samples at 1 μM concentration (only NU was analyzed at 2 μM conc.) in the selected K⁺- (a) and Na⁺-rich (b) buffer solutions, run at 80 V at room temperature (r.t.) for 2 h in Tris-Borate-EDTA (TBE) 1X buffer; lane 1: NU; lane 2: NU172; lane 3: cycNU172-EG2; lane 4: cycNU172-EG3; lane 5: cycNU172-Ph; lane 6: cycNU172-Pro.

Figure 5

Enzymatic resistance experiments performed on NU172 and its cyclic derivatives incubated in 80% fetal bovine serum (FBS) as monitored by 20% denaturing polyacrylamide gel electrophoresis up to 72 h (time points: 0, 0.2, 0.5, 1, 2, 3, 5, 24, 48 and 72 h): (a) representative 20% denaturing PAGE (8 M urea) at 1 μM sample concentration, run at constant 200 V at r.t. for 2.5 h in TBE 1X as running buffer; (b) intensity of each oligonucleotide band on the gel expressed as percentage of the remaining intact aptamer (see legend for detail) with respect to the untreated oligonucleotide, analyzed up to 24 h. Data are reported as mean values ± SD (error bars) for multiple determinations (at least five). Obtained values were also fitted with an equation for first order kinetics (lines), allowing calculation of the half-life in serum of each aptamer (t_1/2).

Figure 6

Coagulation curves of fibrinogen in the presence of thrombin and different anticoagulant agents (NU172, NU and cyclic NU172 analogues, see inset for details), evaluated by means of light scattering experiments in phosphate buffer solution (PBS). All the aptamers were analyzed at a thrombin:aptamer ratio of 1:2.