Guiding the Design of Synthetic DNA-Binding Molecules with Massively Parallel Sequencing

Abstract

Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 1012-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for polyamides containing β/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing 5′-WCGCGW-3′ from 5′-WGCGCW-3′. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications.

Synthetic genome readers target clustered binding sites across diverse chromatin states

“…Fluorescence intensity is proportional to the association constant between hairpin polyamide and the underlying DNA sequence (20,21). Sequence specificity can also be determined with NGS by solution-based systematic evolution of ligands by exponential enrichment sequencing (SELEXseq) or Bind-n-sequencing approaches (25,38,39). In the NGS-based approaches, a polyamide with an affinity handle, such as biotin, is incubated with duplex DNA (a library bearing all 10 12 sequence permutations of a 20-bp site).…”

“…We turned to in vitro cross-linking experiments to determine whether the differences between the specificity of 3 and 4 were due to more efficient enrichment of high-affinity sites, as is often observed in SELEX experiments (25). In cross-linking experiments, we have previously shown that 4 displays higher specificity for a sequence with a single base-pair mismatch, 5′-AAGAGGAAG-3′ than a sequence with double base-pair mismatch to the seed motif 5′-AAGAGGAGG-3′ (31), where the locations of the mismatches are highlighted.…”

“…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

“…For the large-scale analyses of DNA sequence specificities, we performed Bind-n-Seq 30,31,35 experiments with a next-generation sequencer, Ion PGM, with a library of oligomers containing randomized 21-mer. Polyamides 1-5 were biotinylated for this assay to yield polyamides 6-10 by following a previously reported procedure.…”

“…[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. 1) to investigate the utility of the AO unit with respect to affinity toward a DNA sequence, sequence specificities of DNA binding, aqueous solubilities, and cell permeabilities.…”