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Published October 24, 2012 | Supplemental Material + Published
Journal Article Open

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.

Additional Information

© 2012 American Chemical Society. ACS AuthorChoice. Received: September 6, 2012. Published: September 26, 2012. This work is supported by the National Institutes of Health (GM27681). J.L.M. is supported by a postdoctoral grant from the American Cancer Society (PF-10-015-01-CDD). We thank Adam Urbach for the gift of a precursor to compound 8. We also thank Adam Urbach (Trinity University) and Sarah Lockwood (UC Davis) for helpful discussions. The authors declare no competing financial interest.

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Published - ja308888c.pdf

Supplemental Material - ja308888c_si_001.pdf

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