Fig. 4: Evidence for self-reproduction.

a Two-fragment self-reproduction experimental assay. The ribozymes are first fragmented into two pieces (WXY and Z) at position 145, then incubated at defined time points. The formation of longer covalent copies is monitored by gel electrophoresis. b Two-fragment self-reproduction assay for 5 designs and Azo, assayed for 0, 1, 6 and 24 h. The assayed ribozymes are documented in the table on the right of the gel: the number of mutations from the Azoarcus ribozyme is indicated in their name, and their activity score for the self-splicing assay in the last column. The molecular scale in nucleotides. The red arrows are pointing at the recombined covalent ribozymes. For other designed ribozymes with 15–60 mutations, see Supplementary Fig. 8. c Four-fragment self-reproduction assay for one design and Azo, assayed for 1, 2, 4 and 24 h. The dca.T03.19.1 design and Azoarcus ribozyme are first fragmented into four fragments (W, X, Y and Z) at the positions 62, 99 and 145. The molecular scale in nucleotides. Two conditions were assayed: the four fragments in the absence of the covalent ribozyme; and the four fragments in the presence of the covalent ribozyme (condition +). The red arrow shows the presence of the fully recombined covalent ribozymes. d The dca.T03.19.1 ribozyme. The colors represent the 4 different fragments, and the nucleotides colored with a darker shade highlight the position of mutations compared to the Azoarcus ribozyme.