Extended Data Figure 2: The first two base pairs of the P2 stem in class I DNAzymes are crucial for efficient self-cleavage, whereas the sequence of the P1 stem does not affect cleavage efficiency.

a, b, Left, schematic representation of DNAzyme variants as they would be used at the 5′ (a) or 3′ (b) end of a staple; black letters indicate bases that were classified as highly conserved²⁶, red letters indicate positions that we identified to be crucial for efficient cleaving activity, P1 and P2 indicate the two stems, and black triangles indicate the cleavage sites. Right, agarose-gel-electrophoretic analysis of the reaction kinetics of the respective DNAzyme variant. The slower migrating band corresponds to the uncleaved oligonucleotide and the faster migrating band to the reaction product. The activity time constant τ is defined as the first time point at which the intensity of the product band exceeds the intensity of the uncleaved DNAzyme. c, d, Comparison of the cleavage kinetics of different sequence variations of the DNAzymes shown in a and b, respectively.