Specific down regulation of endogenous gene in <i>P. falciparum</i> using PNA molecules.

<p><b>(A)</b>, 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>(B)</b>, Down-regulation of an essential gene using specific Sec13-PNA molecules reduces parasites' viability. NF54-<i>luc</i> parasites constitutively expressing the <i>luciferase</i> 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 <i>luciferase</i> 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 <i>luciferase</i> reads compared with untreated NF54-<i>luc</i> parasites <b>(C).</b> 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 <b>(D)</b>. 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 <i>lucifearse</i> expression compared with untreated NF54-<i>luc</i> parasites as above. All experiments were done in triplicate and the average is presented with SE.