Practice question

Specificity principle of C2H2 zinc finger modular recognition lies in each finger contacting three base pairs. Structural biology of Zif268-DNA complex by Pavletich and Pabo 1991 revealed that alpha helix of each finger inserts into major groove, amino acid residues at positions -1, 2, 3 and 6 relative to helix start make sequence-specific contacts. Triplet code allows prediction: for example Arg at -1 contacts guanine, Asp at 2 contacts cytosine, pattern learned from SELEX. Therefore three finger protein recognizes 9 bp, four finger 12 bp, six finger 18 bp. Context dependence exists because neighboring fingers influence docking, sometimes overlapping fourth base cross-strand contact extending recognition beyond three bases, but canonical model simplifies design. To achieve unique genomic address in rice genome ~430 Mb, 12 bp occurs ~250 times, 18 bp statistically once, but paired ZFNs double site to 18-36 bp effectively unique plus dimer requirement increases specificity reducing off-target cleavage versus restriction enzymes recognizing 6 bp. Modular assembly libraries of 64 triplet-specific fingers enable construction of ZFN targeting almost any GNNGNNGNN sequence, though AT-rich sequences remain challenging requiring selection approaches.