Figure 1.

Scheme for the in vitro selection, modified after (18). The double-stranded library DNA contained a T7 promoter, a hammerhead ribozyme, and 150 randomized positions flanked by constant regions. (A) Transcription by T7 polymerase led to (B) co-transcriptional self-cleavage of the hammerhead ribozyme and generated the RNA library with 5′ hydroxyl groups. (C) Incubation of these RNAs with cTmp, Yb³⁺, and 50 mM Tris/HCl pH 8 allowed some RNAs to catalyze the nucleophilic attack of their 5′-hydroxyl group onto cTmp, and thereby self-triphosphorylate. (D) Incubation with the R3C ligase ribozyme (41) covalently linked 5′-triphosphorylated RNAs to biotinylated oligonucleotides. (E) Biotinylated sequences were isolated with streptavidin-coated magnetic beads. The selected sequences were then (F) reverse transcribed and (G) PCR amplified to regenerate the double-stranded DNA library, now enriched for sequences catalyzing self-triphosphorylation.