Assembly of chimeric RNA constructs. (A) Outline of the procedure. Regulatory RNAs contained a 5′ biotin, two SREs (SRE+ = WT or SRE−, that is, with an inactivating point mutation in each SRE) and a poly(A) sequence, which was protected against deadenylation by an N40 sequence at the 3′ end. Alternatively, regulatory RNA was used that carried a poly(A) sequence but no SREs (not shown). The regulatory RNA was hybridized to immobilized oligo(dT), then divalent streptavidin and the capped and 3′-biotinylated nLuc reporter RNA were added in a stepwise manner. The chimeric construct was eluted in low-salt buffer. The scheme shows the assembly of forward constructs. Flipped constructs were assembled in the same manner, but 3′-biotinylated regulatory RNAs were used. For a full description of the procedure, see Materials and Methods. (B) Assembly of chimeric RNAs assayed by native gel electrophoresis. Chimeras [forward-SRE±p(A)] were assembled as described in panel A and Materials and Methods. The figure shows the analysis of input and product RNAs by electrophoresis through a 5% nondenaturing polyacrylamide gel. Lanes 1–3 display the input RNAs as indicated. Lanes 4–6 demonstrate the retardation of input RNAs upon mixing with divalent streptavidin (STV). Two retarded bands in one lane are presumably due to binding of one or two RNAs to one molecule of streptavidin. Lanes 7, 8 show the flow-through of the oligo(dT) matrix after loading with regulatory RNAs; binding was essentially complete. Lanes 9 and 10 show the flow-through of excess nLuc RNA in the last assembly step. “nLuc+”′ and “nLuc−” refer to the nLuc RNAs from the assembly reactions with SRE+ and SRE− regulatory RNAs, respectively. Lanes 11 and 12 show the eluate in formamide-containing loading buffer without heat denaturation. The main band (arrowhead) is a novel species that we interpret as the desired chimeric RNA. Upon heat denaturation (lanes 13, 14), the input RNAs are restored. Residual amounts of input RNAs can be detected: Based on a comparison to other lanes in this gel, numbered bands are (1) a complex between one regulatory RNA and streptavidin, (2) bare nLuc RNA, and (3) a complex between two regulatory RNAs and streptavidin. The broken line indicates the upper end of the gel.
