Preparation of Homogeneous HIV-1 RNA

The UTR:ΔDIS:A34U construct used in this work is derived from the first 356 nt of the HIV-1 NL4-3 isolate cloned into a pUC19 parent plasmid. Both the ΔDIS (replacement of the SL1 palindromic loop with a GAGA tetraloop) and A34U mutations prevent genomic dimerization and facilitate homogeneous RNA preparation (Skripkin et al. 1994 (link); Helga-Maria et al. 1999 (link); Andersen et al. 2004 (link)). The final construct size, with mutations is 352 nt. The transcription template was generated by digestion of pUC19-UTR:ΔDIS:A34U plasmid with FokI (New England Biolabs). RNAs were prepared via in vitro transcription with T7 RNA polymerase (Milligan et al. 1987 (link)) and purified using 8 M urea (denaturing) polyacrylamide gel electrophoresis (PAGE). Desired bands were excised, crushed, and soaked in RNA elution buffer (0.5 mM NH₄OAc, 1 mM EDTA) overnight at 37°C. Eluted RNA was butanol extracted, ethanol precipitated, and resuspended in diethylpyrocarbonate (DEPC)-treated water. Purified RNA was folded in 50 mM HEPES (pH 7.4) buffer by heating at 80°C for 2 min, cooling to 60°C for 2 min, adding 1 M MgCl₂ to a final concentration of 1 mM, incubating at 37°C for 30 min, and cooling on ice for 30 min. Different durations of the 37°C incubation step were tested for optimal sample homogeneity (data not shown).