Selection scheme designed to retain sequence variation all the way to the 3′ end of RNA transcripts. (A) Pool RNA tethered to pRpp was incubated with 4SUra. Active sequences react to form tethered 4-thiouridine. (B) Active sequences were purified away from unreactive sequences, using an APM gel. (C) Using T4 RNA ligase, purified ribozymes were then ligated to an adenylated DNA oligo containing an EarI restriction site and 3′ RT-PCR primer binding region. (D) RT-PCR was performed to amplify ribozyme DNA sequence and add a T7 promoter (T7) to the 5′ end of the sequences. (E) Digestion with EarI-generated DNA sequence lacking a fixed 3′ primer-binding site, which was then transcribed to generate RNA for another round of selection.
