An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins

Department of Genetics, Development and Cell Biology, Bioinformatics and Computational Biology Program, Iowa State University, Ames, IA 50011, USA.
Nucleic Acids Research (Impact Factor: 9.11). 01/2009; 37(2):506-15. DOI: 10.1093/nar/gkn962
Source: PubMed

ABSTRACT Zinc-finger proteins (ZFPs) have long been recognized for their potential to manipulate genetic information because they can be engineered to bind novel DNA targets. Individual zinc-finger domains (ZFDs) bind specific DNA triplet sequences; their apparent modularity has led some groups to propose methods that allow virtually any desired DNA motif to be targeted in vitro. In practice, however, ZFPs engineered using this 'modular assembly' approach do not always function well in vivo. Here we report a modular assembly scoring strategy that both identifies combinations of modules least likely to function efficiently in vivo and provides accurate estimates of their relative binding affinities in vitro. Predicted binding affinities for 53 'three-finger' ZFPs, computed based on energy contributions of the constituent modules, were highly correlated (r = 0.80) with activity levels measured in bacterial two-hybrid assays. Moreover, K(d) values for seven modularly assembled ZFPs and their intended targets, measured using fluorescence anisotropy, were also highly correlated with predictions (r = 0.91). We propose that success rates for ZFP modular assembly can be significantly improved by exploiting the score-based strategy described here.

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    • "Despite the increasing experience gained with ZFNs and until most recent findings were reported [77], the targeting capacity (the diversity of sequences that can be recognized) of ZFNs was considered to suffer limitations [78] [79] [80]. TALENs appear to be less subject to these constraints due to the greater simplicity of their DNA-binding domains. "
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    • "This ZFN was named ChR1-R. The complementary ZFN ChR1-L was assembled from three ZF-domains that were expected to identify three GNN nucleotide triplets (Ramirez et al., 2008; Sander et al., 2009). Since the performance of such an assembled array was unknown, we created two ChR1-L versions named ChR1-L1 and -L2. "
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    • "In a previous report, the zinc finger module for 5'-GAC-3', with the same amino acid sequences we used in this study, was assembled into several 3-zinc finger proteins and they showed a DNA binding ability to the expected target sites independent of the examined neighboring zinc finger modules [18; 21]. In addition, an additive DNA binding affinity is expected from the recently reported affinity-based scoring scheme [18] by an assembly of the zinc finger module for 5'-GAC-3'. The observed decrease in DNA binding suggests that a context-dependent reduction in the DNA binding occurred by assembly of the zinc finger module for 5'-GAC-3', though the finger itself has the potential to recognize the triplet [21; 22]. "
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