Structure and specificity of the I-AniI LAGLIDADG HE. (a) The I-AniI endonuclease (shown here, pdb code 2qoj) was used in this study, with the addition of activating mutations—Y2, M4 and M5, detailed in the methods—identified in previous work (32). Monomeric LAGLIDADG endonucleases are pseudo-symmetric, with two enzyme halves binding to the left (– half) and right (+ half) sides of the DNA target that flank the central four bases where cleavage occurs (arrow). The N-terminal domain binds to the (–) half-site and the C-terminal domain binds to the (+) half-site. The linker between the two regions is shown in red and the termini are marked with pink (N) and orange (C) spheres. The goal of our work is to alter the target site substrate preferences of these enzymes in order to direct their cleavage to genomic sites of interest. Many new variants cleaving single base-pair substitutions in the I-AniI target are presented in this work, and the labeling scheme for these variants is presented here. The ‘Selected’ enzymes were identified from fully randomized libraries, ‘Computationally guided’ enzymes were either improved versions of previous computational designs or selected from libraries containing computationally identified motif contacts, and the ‘Previously published’ enzymes are presented as well to show the full range of currently targeted positions in the I-AniI interface. (b) Experimentally determined specificity for the I-AniI endonuclease (Y2), derived from previously published kinetic data on each single base-pair substitution (12). Experimental specificity is defined in the Methods section on computational specificity prediction—a value close to 1.0 indicates that the enzyme has high specificity and a value of 0.25 indicates that all nucleotides are cleaved equally or that one other nucleotide is significantly preferred over the target nucleotide.