Specificity landscapes of DNA binding molecules elucidate biological function

Abstract

Evaluating the specificity spectra of DNA binding molecules is a nontrivial challenge that hinders the ability to decipher gene regulatory networks or engineer molecules that act on genomes.Here we compare the DNA sequence specificities for different classes of proteins and engineered DNA binding molecules across the entire sequence space. These high-content data are visualized and interpreted using an interactive "specificity landscape" which simultaneously displays the affinity and specificity of a million-plus DNA sequences. Contrary to expectation, specificity landscapes reveal that synthetic DNA ligands match, and often surpass, the specificities of eukaryotic DNA binding proteins. The landscapes also identify differential specificity constraints imposed by diverse structural folds of natural and synthetic DNA binders. Importantly, the sequence context of a binding site significantly influences binding energetics, and utilizing the full contextual information permits greater accuracy in annotating regulatory elements within a given genome. Assigning such context-dependent binding values to every DNA sequence across the genome yields predictive genome-wide binding landscapes (genomescapes). A genomescape of a synthetic DNA binding molecule provided insight into its differential regulatory activity in cultured cells. The approach we describe will accelerate the creation of precision-tailored DNA therapeutics and uncover principles that govern sequence-specificity of DNA binding molecules.

Synthetic genome readers target clustered binding sites across diverse chromatin states

“…CSI studies demonstrated that affinities and specificities of polyamides for their cognate sites rival the affinities and specificities of natural transcription factors (20)(21)(22)(23). Moreover, CSI data yielded polyamide affinity measurements for every 10-mer sequence that occurs in any given genome (21). Applying this information to map binding potential across the genome led to CSI-based "genomescapes" that predict thousands of putative polyamide-binding sites of varying affinities across the human genome (21,22).…”

“…In particular, we developed cognate site identifier (CSI) analysis to interrogate DNA sequence specificity and affinity comprehensively for any given sequence across half a million permutations of a 10-mer binding site (20,21). CSI studies demonstrated that affinities and specificities of polyamides for their cognate sites rival the affinities and specificities of natural transcription factors (20)(21)(22)(23). Moreover, CSI data yielded polyamide affinity measurements for every 10-mer sequence that occurs in any given genome (21).…”

“…To understand the rules that govern polyamide function, several groups have independently examined the DNA sequence specificity of this class of molecules in vitro (20)(21)(22)(23)(24)(25). In particular, we developed cognate site identifier (CSI) analysis to interrogate DNA sequence specificity and affinity comprehensively for any given sequence across half a million permutations of a 10-mer binding site (20,21). CSI studies demonstrated that affinities and specificities of polyamides for their cognate sites rival the affinities and specificities of natural transcription factors (20)(21)(22)(23).…”

“…Remarkably, rationally designed polyamides fed to Drosophila larvae induced classic homeotic patterns of developmental reprogramming (19). To understand the rules that govern polyamide function, several groups have independently examined the DNA sequence specificity of this class of molecules in vitro (20)(21)(22)(23)(24)(25). In particular, we developed cognate site identifier (CSI) analysis to interrogate DNA sequence specificity and affinity comprehensively for any given sequence across half a million permutations of a 10-mer binding site (20,21).…”

Sequence-specific DNA binding by long hairpin pyrrole–imidazole polyamides containing an 8-amino-3,6-dioxaoctanoic acid unit

“…[25][26][27][28][29] Sequence specificities of Py-Im polyamides have also been investigated by using high-throughput sequencing. [30][31][32] Here, we designed and synthesized thirteen Py-Im polyamides ( Fig.…”