Figure 2:

The process for adding type P1 and P3 Twister ribozymes along with the ribozyme sequences are shown. The type P1 Twister ribozyme sequence (cyan) has a ribozyme stem sequence (red) between its cleavage site (black arrow head) and 5’ end. The type P3 Twister ribozyme sequence (blue) has a ribozyme stem sequence that is between its cleavage site and 3’ end.

The ligation sequences (orange), which are unique for each ligation site are designed so that they will not form helices with ligation sequences from other ligation sites. They are also designed to include the 5’ and 3’ tRNA intermediate sequences (orange with green outline).

The type P1 Twister is added to the upstream transcript (labeled “RNA 1” and green) by replacing the ribozyme’s stem sequence with the 3’ ligation sequence. Subsequently, the type P3 ribozyme Twister sequence is added to the downstream transcript (labeled “RNA 2” and green) by replacing its ribozyme stem sequence with the 5’ ligation sequence. If the 5’ tRNA intermediate sequences have been modified to avoid erroneous hybridization, then mutations need to be made that maintain the helices within both ribozymes. The sites to make these mutations (blue or cyan with red outline) are the same for each ligation site, but the exact mutations may differ.

After cleavage of both ribozymes, the 5’ and 3’ ligation sequence form a helix that will contain both tRNA intermediate sequences with 2’,3’-cyclic phosphate and 5’-OH groups. This ligation site can be recognized by RtcB, which ligates the 5’ and 3’ overhangs, leading to trans ligation of the two RNAs of interest.