FIG. 3.

Action of recombinant dicer on HDV and other RNAs. Lanes 1 to 12, RNA samples either with (+) or without (−) dicer during incubation for 16 h at 37°C. Each sample contained 1 μg of double-stranded RNA. To each sample was added a ³²P-labeled RNA as follows: lanes 1 and 2, genomic HDV RNA; lanes 3 and 4, antigenomic RNA; lanes 5 and 6, PSTVd RNA; lanes 7 and 8, ASBVd RNA; and lanes 9 and 10, modified genomic HDV RNA. For lanes 11 and 12, we added 1 μg of modified genomic RNA that was not labeled. The modification, as described in the text and in the legend for Fig. 4, was to replace some HDV sequences with those of the predicted precursor to miR-122. (A) Aliquots of each sample were analyzed in nondenaturing gels of 3% agarose followed by ethidium bromide staining and detection by digital imaging. (B) Aliquots were analyzed in gels of 15% polyacrylamide-7 M urea, after which the RNAs were electrotransferred to a nylon membrane. In lanes 11 and 12, miR-122 sequences were detected by low-stringency hybridization using a radiolabeled oligonucleotide probe. ³²P was detected by use of bioimager. p, 1-kb DNA ladder; q, 21-bp RNA.