Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome

Sztuba-Solinska, Joanna; Teramoto, Tadahisa; Rausch, Jason W.; Shapiro, Bruce A.; Padmanabhan, Radhakrishnan; Le Grice, Stuart F. J.

Selective 2 0 -hydroxyl acylation analyzed by primer extension (SHAPE) Twenty picomoles of RNA was heated at 90 C for 3 min and slowly cooled to 4 C. The volume was adjusted to 150 ml in a final buffer of 50 mM Tris-HCl (pH 8.0), 100 mM NaCl and 5 mM MgCl 2 . Samples were incubated at 37 C for 15 min. Folded RNA was divided into two equal portions (72 ml) treated with 8 ml of 30mM N-methylisatoic anhydride (NMIA) in anhydrous dimethyl sulfoxide (DMSO) (+) or DMSO alone (À). Tubes were incubated at 37 C for 50 min, and RNA was precipitated at À20 C with 60 ng/ml glycogen, 0.3 M sodium acetate (pH 5.2) and 3 volumes of cold ethanol. Precipitated RNA was collected by centrifugation, washed once in 70% ethanol and resuspended in 10 ml of water. Five picomoles of Cy5-labeled (for NMIA-modified samples) or Cy5.5-labeled (for unmodified samples) primer S720 or S440 (Supplementary Table S1) in 7 ml H 2 O was annealed to the RNA at 85 C for 1 min, 60 C for 5 min and 35 C for 5 min. RNA was reverse transcribed at 50 C for 20 min with 100 U reverse transcriptase (RT) (Invitrogen superscript III), 1Â RT buffer (Invitrogen), 5 mM dithiothreitol (DTT) and 500 mM dNTPs (Promega). RNA was hydrolyzed with 200 mM NaOH (4 M) for 5 min at 95 C, and reactions were neutralized with an equivalent volume of HCl (2 M). Sequencing ladders were prepared using the Epicentre cycle sequencing kit according to the manufacturer's instructions and primers labeled with WellRed D2 and LicorIR-800 dyes. Modified and control samples were mixed with the sequencing ladders, precipitated as described earlier in the text, dried and resuspended in 40 ml of deionized formamide. Primer extension products were analyzed on a Beckman CEQ8000 Genetic Analysis Systems as described previously (19) . Electropherograms were processed using the SHAPEfinder program, following the software developer's protocol and included the required pre-calibration for matrixing and mobility shift for each set of primers as described (20, 21) . Briefly, the area under each negative peak was subtracted from that of the corresponding positive peak. The resulting peak area difference at each nucleotide position was then divided by the average of the highest 8% of peak area differences, calculated after discounting any results greater than the third quartile plus 1.5Â the interquartile range. Normalized intensities were introduced into RNAstructure version 5.3 (22) .