Fig. 3.

Effect of chemical modification of the aptamers on their activity. (A) Estimation of critical region possessing activation potency. The predicted secondary structure of the c37 aptamer and its truncated variants were shown (Left). Activation potency of the truncated aptamers was examined. The values were expressed as RLU to the 2.5 μM PSB1114 level without aptamer (Right). (B) Effect of the chemical modification of aptamer. The predicted secondary structure of the aptamer c37_8-40 is shown (Left). The unpaired 2′-fluoro-uridine and 2′-deoxy-adenosine located in the loop structure of c37_8-40, denoted by arrowheads, was modified with a hydroxy (2′OH) and methoxy group (2′OMe), respectively. Activation potency of the modified aptamers was measured and expressed as RLU to the 2.5 μM PSB1114 level (Middle). Inhibitory potency of the aptamers against 100 nM PSB1114 was expressed as RLU to the 100 nM PSB1114 level without aptamer as in A (Right). (C) Agonist and antagonist assay of aptamers to the mutant receptors. Activation potency of the modified aptamers to Y114F and F261A mutant receptors was shown as RLU to the 2.5 μM UTP (Left). Inhibitory activity of the indicated aptamers to each type of P2RY2 stimulated by UTP at the indicated concentrations was expressed as RLU to the indicated concentrations of UTP without aptamer in each type of P2RY2 (Right). Data represent the mean ± SD (n = 3 independent experiments). EC₅₀ and IC50 values obtained from chemical agonists and antagonist were indicated.