P. falciparum Culture
3D7 and Dd2 P. falciparum malaria parasites (MRA-102, 156, MR4, ATCC® Manassas, Virginia) were cultured in human type O+ erythrocytes in complete medium (RPMI 1640, 10 mg/ml Gentamicin (Gibco), 1.36 g/l Hypoxanthine (Acros), 1 M HEPES (Sigma), 7.5% Sodium Bicarbonate (Gibco), 20% Glucose (MP Biomedical), 1 M NaOH (Sigma), 20% Albumax (Gibco), 5% human serum) as previously described . Cultures were maintained in 25-cm2 flasks (Corning) at a volume of 10 ml and were gassed for 30 s with an environment of 3% CO2, 1% O2, and 96% N2, then incubated at 37°C. Synchronization of culture was achieved through sorbitol lysis of mature stage using 5% sorbitol (Fisher) fine-tuned by another lysis 8 hours later .
Microwell plates already containing a library of RNA-probes were diluted in DMSO to 10 mM. A screening of 125 different RNA-probes were performed by diluting infected erythrocytes to 0.025% hematrocrit with a 6% parasitemia into optical bottom 96-well assay plates (Costar #3614, Corning, NY) containing 240 μl of complete medium. RNA-probes were added to a final concentration of 1 μM, 5 μM, 7.5 μM, and 10 μM with a total DMSO level of 0.5%. Plates were incubated in the dark for 30 minutes at 37°C. During microscopic analysis, the BD Pathway HT (BD Biosciences Bioimaging, Rockville, MD) temperature was regulated at 37°C. Each well was fluorescently imaged using fluorescence combinations of Semrock (Rochester, NY) DAPI, CFP, GFP, YFP and Texas Red BrightLine filter sets. We found that 5 μM was the optimal working concentration.
RNA versus DNA specificity solution assay
DNA was extracted from P. falciparum using an adapted phenol/chloroform extraction. Infected erythrocytes were harvested and brought to a 50% hematocrit in PBS. Samples were incubated with cell lysis solution (Promega #A7933, Madison, WI) for 10 minutes at room temperature, followed by centrifugation. After removing the supernatant, pellet was incubated at 55°C for an hour in lysis buffer containing 4 M guanidine HCl (Promega), 10% SDS (Promega), and 20 mg/ml Proteinase K (New England Biolabs), and left overnight at 4°C. DNA was then extracted using Phenol/chloroform/isoamyl alcohol (Sigma) following the standard procedures.
RNA was extracted from infected erythrocytes with Trizol LS (Life Technology), as previously described in Le Roch et. al. 2003 .
To scan fluorescence emission, the SpectraMAX GeminiEM (MDS Analytical Technologies, Toronto, Canada) and the SoftMax Pro program were used. Using black, clear bottom, 96-well assay plates (Costar #3904), 10 two-fold serial dilutions were performed with extracted DNA and RNA starting at 200 μg/ml. Steady state concentrations of RNA or DNA at 10 μg/ml, were mixed with two-fold serial dilutions of the opposing nucleic acid. 132A, 107E, and 107F RNA probes were added to wells at a 10 μM concentration. Endpoint readings with an excitation of 500 nm, cutoff at 515 nm and emission of 605 nm for 132A, and excitation of 435 nm, cutoff at 605 nm and emission of 610 nm for 107E and 107F. DNA and RNA concentration as a function of fluorescence intensity was plotted with SigmaPlot.
Synchronized infected erythrocytes were stained with nuclear dye DAPI (Molecular Probes #D21490, Eugene, OR) or Hoechst 34580 (Molecular Probes #H21486, Eugene, OR) diluted in H2O and added to reach a final concentration of 20 ng/μl or 5 μg/ml respectively. RNA specific dyes were then added to reach a concentration of 5 μM and plates were incubated with both dyes in the dark at 37°C for 30 minutes. Images were taken using the BD Pathway HT with the temperature regulated at 37°C. A macro, an instruction set for the Pathway HT, was designed to take images of the same frame using the DAPI filter wheel, then the corresponding filter for RNA dye, then transmitted light, auto focusing for each image, moving to another frame in the same well, and to repeat in subsequent wells.
Synchronized infected erythrocytes were diluted with uninfected erythrocytes in complete media (i.e. 0%, 1.5%, 3%, 4.5%, 6%). Diluted probes were added to reach a final concentration of 5 μM, incubated in the dark for 30 min at 37°C and the BD Pathway HT measured fluorescence intensity. Data analyses were performed using Microsoft Excel 2004 for Mac. Data points were graphed by 1) calculation of the mean of triplicates per sample condition, 2) subtraction of the fluorescence background, and 3) conversion to relative fluorescence units.
Quantitative assay evaluation
Three 96-well microplates with parasite-infected erythrocytes were used as a positive control, and three microplates with uninfected erythrocytes were used as a negative control. All plates were diluted to a 0.025% hematocrit, and 6% parasitemia. 107E, 107F, and 132A dyes were added to microplates and the Pathway HT determined fluorescence intensity. The formula z' = 1 - (3SD+ + 3SD-)/|Ave+ - Ave-| was used to caculate the Z' value. Where SD+ represents the positive control standard deviation, SD- the negative control standard deviation, Ave+ the mean value of the positive control, and Ave- the mean value of the negative control.
3D7 parasite cultures were incubated with 0.1 nM, 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM, and 500 nM concentrations of chloroquine or artemisinin (Sigma #C6628-25G, St. Louis, MO) for 72 hours. Dd2 parasite cultures were incubated with 5 nM, 25 nM, 75 nM, 100 nM, 150 nM, 200 nM, 300 nM, and 500 nM concentrations of chloroquine. RNA dyes were added to reach 5 μM final concentration and incubated in the dark for 30 minutes at 37°C. Microscopic analysis was performed at a regulated temperature of 37°C. Montage images of 4 by 4 frames were acquired with transmitted light and the corresponding filter for RNA dyes. Images were merged to verify infected erythrocytes by having the fluorescent target overlap with visible hemazoin in transmitted light images. The Pathway HT calculated the parasitemia by using the Region of Interest (ROI) function to count fluorescent parasites. Parasitemia was determined by manual counts of fluorescent images and Giemsa-stained blood smears, and compared to Pathway HT counts. Samples of identical cultures were taken to perform a SYBR Green assay as previously described . Data were analyzed using Microsoft Excel for Mac, and graphs were plotted using SigmaPlot 10 (Systat).