(W1430-01-01) Development of Physicochemical Profiling Platform for Therapeutic Oligonucleotides V Biological Function Analysis of Oligodeoxynucleotides

Purpose: To develop oligonucleotide-based pharmaceuticals, it is important to understand the relationship between their physical properties and biological activities. In this study, we focused on evaluating the antithrombin activity and affinity for thrombin of thrombin aptamer (TBA) forming a guanine quadruplex (G4) structure, and four mutated TBAs with partial sequence modification as model oligodeoxynucleotides.

Methods: TBA and four mutated TBAs (T1, T2, T3, and T4) were designed to control the higher-order structures of TBA forming G4 structures (Table 1). TBAs were dissolved in phosphate-buffered saline (PBS) to mimic the potassium and sodium concentrations inside the cells and body fluids (PBS_in and PBS_out, respectively) and heated at 95 °C for 5 min, followed by cooling to 25 °C at a rate of 1 °C /min to form the G4 structure. To measure antithrombin activity, the sample solutions were mixed with thrombin solution followed by fibrinogen solution, and turbidity measurements were performed at a wavelength of 380 nm. The coagulation time was defined as the time with the highest coagulation rate, which was determined by differentiating the time-absorption curve. The interaction between thrombin and TBAs was evaluated using an amplified luminescent proximity homogeneous (alpha) assay. Thrombin was immobilized with unconjugated-acceptor beads of alpha assay by Borch reaction via aldehyde group on the beads surface and amino group of thrombin. The 5’-terminal biotinylated probe A (5’-AGAATGCTGAGATGTAGA-3’) and TBAs with a probe A complementation sequence at the 3’-end were mixed and hybridized at 25°C for 10 min. Hybridized oligodeoxynucleotides were mixed with thrombin-immobilized acceptor beads in PBS_in or PBS_out containing 0.5 %Triton X-100 and 0.1 % bovine serum albumin, pipetted into the 96 wells plate, and incubated at 25°C for 1hour. Subsequently, streptavidin-coated donor beads were added and incubated at 25°C in the dark for 1 h. The 520-620 nm emission output signal from the acceptor beads was measured using an EnSight plate reader with a 680 nm laser to excite the donor beads.

Results: TBA interacts with positively charged exosite I and II regions of thrombin, and then inhibit thrombin anticoagulant activity. Longer coagulation times result from the higher anticoagulant activity of TBAs. As shown in figure 1, the anticoagulant activity of TBA was higher in PBS_out than that in PBS_in. In the comparison of mutated TBAs, T2 and T3 showed anticoagulant activity in PBS_in and PBS_out; however, no anticoagulant activity was observed for T1 and T4 at concentrations up to 40 nM. T1 showed modest anticoagulant effects only in PBS_in and not PBS_out at sub-M concentrations. An alpha assay was performed to determine the interactions between thrombin and TBAs. As shown in figure 2, the equilibrium dissociation constant for TBA was sub-nM in both PBS_out and PBS_in, and the lowest value among the TBA and mutated TBAs. This indicated that TBA had the highest affinity for thrombin. While the mutants T2 and T3 showed nanomolar order equilibrium dissociation constants for thrombin, T1 bound slightly to thrombin only in PBS_in, and no binding of T4 was observed. This is consistent with the anticoagulant activity test results.

Conclusion: TBA, T2, and T3, which form two planar guanine(G)tetrads, showed high antithrombin activity. In contrast, mutants T1, which forms three planar G-tetrads, and T4, which does not form a G-quadruplex structure, show no antithrombin activity. The binding affinity of the respective TBAs to thrombin, measured by alpha assay, also showed the same trend as the anti-thrombin activity. CD and NMR analyses have shown that T2 and T3 have a topological structure similar to that of TBA, while T1 forms a different topology to TBA. These results indicate that the topology of G-quadruplex structure-based aptamers is involved in the biological functions of TBAs.

Acknowledgements: This study was conducted as part of Center of Excellence for Pharmaceutical Materials Science (Materials Open Platform for Pharmaceutical Science) led by National Institute for Materials Science.

Table 1 Oligodeoxynucleotide sequences used in this study

Figure 1 Concentration dependency of anticoagulant activity of thrombin aptamers.

Figure 2 Saturation isotherms of thrombin aptamers to human thrombin