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 10(12)-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.

Figure 1

(A) Hairpin Py-Im polyamide recognition of the DNA minor groove. Aromatic amino acid ring pairs recognize distinct DNA base pairs. Complete structures for each hairpin polyamide-biotin conjugate used can be found in Supporting Information. (B) Scheme for Bind-n-Seq analysis of DNA binding polyamides. Double-stranded DNA containing a degenerate, 21-bp segment is enriched, purified, and analyzed via high-throughput sequencing. Commonly bound DNA consensus sequences are identified via motif searching.

Figure 2

Structures of polyamides 1–6 and primary motifs identified for each via high-throughput analysis.

Figure 3

(A) Polyamide 4 with β/Im pairs as found in the forward (left) and reverse (right) binding orientations. (B) Second generation molecules used to probe the effect of turn modification (7, 8) on polyamide-DNA binding preferences.

Figure 4

High-throughput sequencing guided redesign of polyamide 4.

Figure 5

High-throughput sequencing guided redesign of turn unit for polyamide 3 with a single β/Im pair.