Sir Aaron Klug

The Use of Zinc Finger Peptides for the Regulation of Gene Expression


Abstract

Zinc fingers are small peptide motifs that bind specifically to three successive base pairs of the DNA double helix. These motifs can therefore be used as modular building blocks for the construction of larger protein domains that recognize longer specific DNA sequences. We have used a technique called phage display to create a large library (or repertoire) of different zinc fingers from which selections are made for binding to a given DNA sequence triplet. From this database there have been elucidated elements of recognition rules that relate the amino acid sequence of a finger motif to its preferred DNA binding site.

Control of gene expression, using such specifically selected zinc finger peptides has been demonstrated by the specific inhibition of an oncogene in a mouse cancer cell line and also, conversely, by activating particular genes in expression plasmids. These experiments demonstrated that zinc-finger DNA binding domains can be engineered de novo to recognize given DNA sequences.

Five to six individual zinc fingers linked together would recognize a DNA sequence 15-18 basepairs in length, sufficiently long to constitute a rare address in the human genome. By adding functional groups to the engineered DNA binding domains, eg silencing or activation domains, novel transcription factors can be generated to up- or downregulate expression of a target gene. Some recent applications will be described:

(i)the disruption of the infective cycle of infection by herpes simplex virus
(ii)activating the expression of erythropoietin (EPO) in a human kidney cell line
(iii)activating the expression of vascular endothelial growth factor (VEGF) in a human cell line.


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