Figure 5

Identification of catalytic RNA from a genomic library. (a) Preparation of the genomic library. Genomic DNA is first partially digested and fragments of approximately 150 bp (blue) are gel-purified and incubated with Taq polymerase to give them 3' A overhangs. In the next step, ligation of covalently closed oligonucleotides (yellow and purple) to the library prevents the unwanted combination of DNA fragments. After removal of DNA hairpins, a T7 promoter (magenta) is then added by PCR, yielding an amplified linear library. (b) The in vitro selection scheme. The library is further amplified by PCR using a 5'-phosphorylated reverse primer and a biotinylated forward primer that allows the isolation of the phosphorylated strand using streptavidin beads. Single strands are individually circularized by ligation with a splint oligonucleotide and the second strand is added by incubation with Taq polymerase and deoxynucleoside triphosphates. The resulting nicked double-stranded library is suitable for rolling-circle transcription by T7 polymerase [98], yielding multimeric RNA species potentially encoding sites of self-cleavage (red triangles). The RNA is then incubated for self-cleavage, and active molecules (dimers) are size-selected. The scheme is completed by preparation of the next-generation DNA library using reverse transcription-PCR (RT-PCR). Modified from [29].