Figure 2.

Self-cleavage scheme. Plasmid DNA from individual clones or the enriched DNA pool after the last selection round were amplified by PCR and immobilized on streptavidin beads. The two individual DNA strands were recovered by treatment with concentrated ammonia under different conditions as described (49) (see also Materials and Methods). The biotinylated strand was used for a primer extension reaction with modified triphosphates 1 and 2 and a 5′-fluorescein (Fl)-labelled primer 1. The resulting double-stranded product was immobilized on streptavidin magnetic beads and single-stranded molecules were eluted by treatment with ice-cold sodium hydroxide. Eluates were neutralized immediately and incubated at 37°C. The RNA sequence following cleavage that was initially unknown is indicated as ‘??????’.