Book of Abstracts: Albany 2011

category image Albany 2011
Conversation 17
June 14-18 2011
©Adenine Press (2010)

Getting a Firm Grip on DNA: Understanding DNA Recognition at the Finger-Finger Interface of Zinc Finger Proteins

Zinc finger nucleases hold tremendous potential for site-specifically editing genomes in a variety of organisms (1). However, their utility is predicated on the ability to efficiently create sequence-specific C2H2 Zinc Finger Proteins (ZFPs) for a wide variety of target sequences. Each zinc finger module typically binds to a 3 bp core DNA element (DNA triplet). Zinc finger modules have been characterized that can specify most of the sixty-four possible DNA triplets. These modules can be assembled into multi-finger zinc finger proteins (modular assembly) that bind extended target sites (9-12 bp), where typically three to four zinc fingers are employed for efficient DNA recognition. However, modularly-assembled ZFPs often show poor specificity presumably due to incompatible specificity determinants at the finger-finger interface (2). To understand the influence of these interactions on DNA recognition, we employed bacterial one-hybrid selections (3,4) to identify groups of amino acid residues at the interface of two-finger modules that specify all sixteen 2 bp interfaces between the two DNA triplets. In total, we could identify two-finger modules that specify >90% of these targets. Further analysis of the selected modules suggests the presence of complex interactions at the finger-finger interface that contribute to context-dependent effects on specificity. These selected finger pairs are functional in vivo, as zinc finger nucleases employing these modules generate targeted lesions in zebrafish. Ultimately, understanding finger-finger interactions will allow the rational design of multi-finger ZFPs for their use as artificial proteins and aid the assignment of specificities for naturally-occurring zinc finger proteins.


  1. F. D. Urnov, E. J. Rebar, M. C. Holmes, H. S. Zhang and P. D. Gregory. Nat Rev Genet 11, 636-646 (2010).
  2. C. L. Ramirez, J. E. Foley, D. A. Wright, F. Muller-Lerch, S. H. Rahman, T. I. Cornu, R. J. Winfrey, J. D. Sander, F. Fu, J. A. Townsend, T. Cathomen, D. F. Voytas and J. K. Joung. Nature methods 5, 374-375 (2008).
  3. X. Meng, M. B. Noyes, L. J. Zhu, N. D. Lawson and S. A. Wolfe. Nat Biotechnol 26, 695-701 (2008).
  4. M. B. Noyes, X. Meng, A. Wakabayashi, S. Sinha, M. H. Brodsky and S. A. Wolfe. Nucleic Acids Res 36, 2547-2560 (2008).

Ankit Gupta1, 2
Amy Rayla1
Ryan G. Christiansen3
Nathan D. Lawson1
Gary D. Stormo3
Scot A. Wolfe1, 2

1Program in Gene Function and Expression
2Department of Biochemistry and Molecular Pharmacology
University of Massachusetts Medical School
Worcester, MA 01605, USA

3Department of Genetics
Washington University School of Medicine
St. Louis, MO 63108, USA

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