Targeted DNA Methylation Using an Artificially Bisected M.HhaI Fused to Zinc Fingers
(A) A schematic of the protein fusions and target DNA sequences indicating the variability in linker length and DNA spacing tested. The linkers connecting the zinc fingers to the N- and C-terminal fragments were varied in 5 amino acid increments (from 0 to 15 amino acids), and combined iteratively. The bases separating the FspI site from the zinc finger binding sites were also varied (0,1,2,3 bases on each side). (B) Truncation of the C-terminus of the C-terminal fragment (indicated in units of amino acids) decreases off-target activity at the methyltransferase. In this experiment X = 3, Y = 1 and Z = 0. The nature of the DNA at site 1 and site 2 (whether a target or non-target site) is depicted at the bottom of the figure and graph. Constructs in which the C-terminus of M.HhaI was truncated by 6 amino acids were used to determine the effect of (C) linker length and (D) target site spacing on methyltransferase activity at the target site. The percent methylation at the target site are indicated in the graphs. All graphs show the mean and the error bar represents the standard deviation of the analysis of plasmid DNA from n ≥3 independent cultures.