Temperature-sensitive protein–DNA dimerizers

Abstract

Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription factors. A hairpin polyamide-YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. Such small molecules function as protein-DNA dimerizers that stabilize complexes at composite DNA binding sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial degree of variability in the linker length is tolerated at lower temperatures. At physiological temperatures, the longest linker tested confers a ''switch''-like property on the protein-DNA dimerizer, in that it abolishes the ability of the YPWM moiety to recruit the natural transcription factor to DNA. These observations provide design principles for future artificial transcription factors that can be externally regulated and can function in concert with the cellular regulatory circuitry.DNA recognition ͉ extradenticle ͉ HOX proteins ͉ polyamide

Defining the sequence-recognition profile of DNA-binding molecules

“…PA2 and PA3 differ solely by a single methylene in the linker connecting the Exd-binding peptide to the polyamide (Fig. 4A) (42). Because PA2 and PA3 are not fluorescently labeled, we detected cognate sites bound cooperatively by these synthetic Hox mimics and Exd, as well as sites bound by Exd alone.…”

Small-molecule regulators that mimic transcription factors

“…Using a novel approach to control ATF activity, Hauschild et al develop a temperature controlled ATF [91]. A hairpin PA was conjugated to a peptide that interacted with the endogenous transcription factor Exd.…”

Controlling gene networks and cell fate with precision-targeted DNA-binding proteins and small-molecule-based genome readers

“…Peptides selected by phage display [133,134], coiled-coiled interactions characterized by protein microarrays [135], as well as small molecules [136], can be attached to ATFs to recruit other natural factors or ATFs. Even short rationally designed dipeptides, such as the ‘WM’ motif used to recruit the Exd (extradenticle) homeodomain, function as IDs (Figures 2B and 2C) [99,114,137]. …”

“…An added layer of temporal and induction level control can be included by using synthetic linkers that enable interactions at physiological temperatures, but are disruptive at higher temperatures [114]. These synthetic temperature-sensitive derivatives can be further elaborated to yield small molecule or secondary metabolite-responsive synthetic IDs.…”

“…Moreover, polyamides can traverse the cell membrane and traffic to the nucleus to exert their effects on gene expression [102-108]. From the induction of striking homoeotic transformations in fruit flies [109], to the inhibition of tumour growth in mice [110,111] and the triggering of the pluripotency network in mammalian cells [112], engineered polyamides can induce remarkable biological outcomes [113,114] (PDB code 3OMJ).…”