Extended Data Figure 4: DNAzyme cassettes comprising two copies of the class I DNAzyme with GT as the first two bases in each P2 stem function robustly and independently of their respective sequences in the context of a long pseudogene.

a, b, Top, schematic representations of two phagemids that contain the same set of staple sequences but are constructed using slightly different DNAzyme architectures. Bottom, analysis of the self-cleavage kinetics of the respective phagemids using alkaline-denaturing agarose gel electrophoresis. Phagemid variant 1 (a) is based on the minimal DNAzyme sequence as identified by us. Phagemid variant 2 (b) is based on the minimal DNAzyme sequence. ref, reference sample containing chemically synthesized staple strands and ‘waste’ DNAzyme snippets. c, Auto-levelled (left) and highly oversaturated (right) scans of a denaturing urea-polyacrylamide gel. L, double-stranded DNA ladder; DZ 1 and DZ 2, chemically synthesized oligonucleotides with the sequence of excised DNAzyme cassettes (84 bases long; DZ 1 and DZ 2 denote different sequences in the P1 and P2 stem regions); chem, mixture of all chemically synthesized staple oligonucleotides needed to fold the pointer (staple lengths vary between 41 and 80 bases, thus multiple bands); bio, mixture of cleaved staple pseudogenes that contain all of the staples needed to fold the pointer, purified either by anion exchange chromatography (IEX) or by ethanol precipitation (EtOH).