FIG. 2.

Cleavage of short RNA oligonucleotides hybridized to various DNA oligonucleotides. The sequences of the RNA and DNA oligonucleotides are given in Fig. 1. The RNA oligonucleotides were 5′ end labeled with T4 polynucleotide kinase. About 20,000 cpm of the ³²P-labeled RNA or capped RNA templates were hybridized to approximately 20 ng of the individual oligonucleotides as described above in the presence of 50 mM Tris-HCl (pH 8.0)–50 mM NaCl–5 mM MgCl₂–2.0 mM dithiothreitol–100 μg of acetylated bovine serum albumin/ml–10 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. The mixtures of RNA and oligonucleotides were heated to 70°C for 10 min and then slowly cooled to room temperature. The reactions were initiated by adding 50 ng of purified wild-type HIV-1 RT and MgCl₂ to a final concentration of 5 mM, in a final volume of 12 μl, and were then incubated at 37°C. Samples were removed at 0.25, 1, 4, and 16 min, and the reactions were terminated by adding 2× RNA loading buffer. The products were heat denatured and separated on a denaturing 15% polyacrylamide–7 M urea gel in Tris-borate-EDTA buffer at 1,600 V for approximately 90 min (7). The gel was dried and autoradiographed.