Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities

Abstract

Designer molecules that can specifically target pre-determined DNA sequences provide a means to modulate endogenous gene function. Different classes of sequence-specific DNA-binding agents have been developed, including triplex-forming molecules, synthetic polyamides and designer zinc finger proteins. These different types of designer molecules with their different principles of engineered sequence specificity are reviewed in this paper. Furthermore, we explore and discuss the potential of these molecules as therapeutic modulators of endogenous gene function, focusing on modulation by stable gene modification and by regulation of gene transcription.

Figure 1

Representation of a TFO binding in the major groove of DNA.

Figure 2

Recognition motifs for TFO-DNA binding. Note the requirement for protonation of cytosine (at N³) in the pyrimidine motif. W-C, Watson–Crick hydrogen bonds; H, Hoogsteen hydrogen bonds; RH, Reverse Hoogsteen hydrogen bonds.

Figure 3

Representation of a hairpin polyamide binding in the minor groove of DNA.

Figure 4

(A) Exemplification of the sequence-specific binding of a hairpin polyamide. (B) Representation of hairpin polyamides with extended DNA target sites. See text for details.

Figure 5

A Cys₂His₂ zinc finger motif showing secondary structure motifs (α, β) and the localization of the conserved cysteine (C) and histidine (H) residues that coordinate the zinc ion, which is depicted by the black sphere. The residues primarily involved with sequence-specific DNA binding are depicted by the squares. The numbers in the squares represent the amino acid positions relative to the first residue of the α-helix.

Figure 6

Polydactyl zinc finger proteins with extended DNA-target sites. (A) Two 3-finger units linked by a canonical linker. (B) Two 3-finger units linked by a flexible linker. (C) Two 3-finger units linked by a structured linker. (D) Three 2-finger units linked by slightly-longer-than-canonical linkers. (E) Two 2-finger units linked non-covalently through dimerization domains.

Figure 7

Mechanisms by which designer DNA-binding agents can modulate gene transcription. (A) Inhibition of transcription elongation by targeting within the transcribed region of a gene. (B) Inhibition or stimulation of transcription initiation by targeting within the regulatory region of a gene. (C) Activation or repression of gene transcription by conjugation to an appropriate effector domain (ED).