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| Status |
Public on Dec 02, 2010 |
| Title |
Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays |
| Organism |
synthetic construct |
| Experiment type |
Other
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| Summary |
C2H2 zinc fingers (C2H2-ZFs) are the most prevalent type of vertebrate DNA-binding domain, and typically appear in tandem arrays (ZFAs), with sequential C2H2-ZFs each contacting 3 (or more) sequential bases. C2H2-ZFs can be assembled in a modular fashion, providing one explanation for their remarkable evolutionary success. Given a set of modules with defined 3-base specificities, modular assembly also presents a way to construct artificial proteins with specific DNA-binding preferences. However, a recent survey of a large number of three-finger ZFAs engineered by modular assembly reported high failure rates (~70%), casting doubt on the generality of modular assembly. Here, we used protein-binding microarrays to analyze 28 ZFAs that failed in the aforementioned study. Most (17) preferred specific sequences, which in all but one case resembled the intended target sequence. Like natural ZFAs, the engineered ZFAs typically yielded degenerate motifs, binding dozens to hundreds of related individual sequences. Thus, the failure of these proteins in previous assays is not due to lack of sequence-specific DNA-binding activity. Our findings underscore the relevance of individual C2H2-ZF sequence specificities within tandem arrays, and support the general ability of modular assembly to produce ZFAs with sequence-specific DNA-binding activity.
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| Overall design |
Protein binding microarray (PBM) experiments were performed for a set of 20 artificial zinc finger arrays (ZFAs). Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. The method is described in Berger et al., Nature Biotechnology 2006.
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| Contributor(s) |
Lam KN, van Bakel H, Cote AG, van der Ven A, Hughes TR |
| Citation(s) |
21321018 |
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| Submission date |
Nov 30, 2010 |
| Last update date |
Jun 10, 2014 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
harm.vanbakel@mssm.edu
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| Organization name |
Mount Sinai School of Medicine
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| Department |
Genetics and Genomic Sciences
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| Lab |
Bakel Lab
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| Street address |
One Gustave L. Levy Place, Box 1498
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platforms (2) |
| GPL11260 |
Agilent custom ME and HK design array [8mer] |
| GPL11261 |
Agilent custom ME and HK design array [9mer] |
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| Samples (80)
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| Relations |
| BioProject |
PRJNA133853 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE25723_RAW.tar |
198.3 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
| Processed data provided as supplementary file |
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