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J Am Chem Soc. 2012 Oct 24;134(42):17814-22. doi: 10.1021/ja308888c. Epub 2012 Oct 10.

Guiding the design of synthetic DNA-binding molecules with massively parallel sequencing.

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1
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.

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.

PMID:
23013524
PMCID:
PMC3483022
DOI:
10.1021/ja308888c
[Indexed for MEDLINE]
Free PMC Article
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