Figure 3. Specific down regulation of endogenous gene in P. falciparum using PNA molecules.

(A), Specific dose dependent down-regulation of PfSec13 expression in parasites incubated with 1.2, 2.4, 4.8 and 9.6 µM Sec13-PNA compared with scrambled PNA sequence (Scr-Sec13-PNA) observed by western blot analysis using antibodies against endogenously tagged PfSec13 (α-HA) and aldolase. (–), no treatment. ScrPNA, Scr-Sec13-PNA. (B), Down-regulation of an essential gene using specific Sec13-PNA molecules reduces parasites' viability. NF54-luc parasites constitutively expressing the luciferase reporter gene were incubated with 1.2, 2.4, 4.8 and 9.6 µM of either Sec13-PNA or Scr-Sec13-PNA. 72h post incubation the parasitemia was counted by direct microscopy (left panel) as well as inhibition of luciferase expression (right panel). The parasitemia decreased in a dose dependent manner only in parasite treated with Sec13-PNA. The gradual increase in inhibition of parasites' viability by Sec13-PNA was calculated as the percentage of luciferase reads compared with untreated NF54-luc parasites (C). The inhibition in growth is correlated with decrease in luciferase expression in parasite treated with Sec13-PNA. R² = 0.99, P<0.05. Dashed line represents 95% confidence interval slope (D). Over time inhibition of parasites viability using PNA. Parasites were incubated with 4.8 µM Sec13-PNA or Scr-Sec13-PNA and luciferase expression was measured 24h, 48h, 72h, and 96h post incubation. The specific reduction in parasites viability is presented as percentage of inhibition of lucifearse expression compared with untreated NF54-luc parasites as above. All experiments were done in triplicate and the average is presented with SE.