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Title: Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays      
dateReleased:
12-02-2010
description:
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. 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.
privacy:
not applicable
aggregation:
instance of dataset
ID:
E-GEOD-25723
refinement:
raw
alternateIdentifiers:
25723
keywords:
functional genomics
dateModified:
05-03-2014
availability:
available
types:
gene expression
name:
synthetic construct
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-25723/E-GEOD-25723.raw.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-25723/E-GEOD-25723.processed.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25723
storedIn:
Gene Expression Omnibus
qualifier:
not compressed
format:
HTML
accessType:
landing page
primary:
true
authentication:
none
authorization:
none
abbreviation:
EBI
homePage: http://www.ebi.ac.uk/
ID:
SCR:004727
name:
European Bioinformatics Institute
homePage: https://www.ebi.ac.uk/arrayexpress/
ID:
SCR:002964
name:
ArrayExpress
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