Polymorphic and Higher-Order G-Quadruplexes as Possible Transcription Regulators: Novel Perspectives for Future Anticancer Therapeutic Applications

Abstract

In the past two decades, significant efforts have been put into designing small molecules to target selected genomic sites where DNA conformational rearrangements control gene expression. G-rich sequences at oncogene promoters are considered good points of intervention since, under specific environmental conditions, they can fold into non-canonical tetrahelical structures known as G-quadruplexes. However, emerging evidence points to a frequent lack of correlation between small molecule targeting of G-quadruplexes at gene promoters and the expression of the associated protein, which hampers pharmaceutical applications. The wide genomic localization of G-quadruplexes along with their highly polymorphic behavior may account for this scenario, suggesting the need for more focused drug design strategies. Here, we will summarize the G4 structural features that can be considered to fulfill this goal. In particular, by comparing a telomeric sequence with the well-characterized G-rich domain of the KIT promoter, we will address how multiple secondary structures might cooperate to control genome architecture at a higher level. If this holds true, the link between drug-DNA complex formation and the associated cellular effects will need to be revisited.

Keywords: G-quadruplex; folding landscapes; gene promoters.

Figure 1

Overview of the solved G4 arrangements of telomeric repeats: (a) antiparallel (PDB 143D); (b) parallel (PDB 1KF1); (c) hybrid 1 (PDB 2HY9); (d) hybrid 2 (PDB 2JPZ); and (e) two-tetrads (PDB 2KKA).

Figure 2

Schematic representation of the proximal promoter of KIT with the G4 structures that can form within this region (kit2: PDB 2KYP; kit*: PDB 6GHO; kit1: PDB 2O3M).

Figure 3

Folding of kit2 as determined in bulk (a) or by single molecule FRET (b) analyses. U refers to the unfolded (or pre-folded) state; I2 to an uncharacterized short-lived intermediate; and G1, G2, and G3 to a kinetically favored, a monomeric, and a dimeric G-quadruplex, respectively.

Figure 4

The two G4 states of kit2. 1K⁺ and 2K⁺ refer to the number of K⁺ ions bound to the folded G4. U refers to the unfolded (or pre-folded) state, and G1 and G2 to the kinetically favored and the structurally solved monomeric G-quadruplex, respectively.

Figure 5

Multiple G4s at the KIT promoter: (a) model of the magnetic tweezer approach used to monitor G4 formation (adapted from [59]) and (b) proposed unfolding pathway of the kit2-kit* fragment (adapted from [60]).

Figure 6

Outline of a pull-down approach leading to the discovery of vimentin as a G4 repeat selective binder (adapted from [68]).