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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    • "To this end, we focused on five different ZF domains (PBSII, Zif268, ZFa, ZFb and ZFc) that were each comprising three fingers with specificity for unique 9 base-pair DNA sequences (22,29–32) (Supplementary Table S1). The selection criteria for choosing these particular ZF domains was as follows: first, the ZF domain should be non-toxic to the host cells (33); second, the ZF domains should be capable of binding orthogonal sequences with high affinity. Based on our estimation of approximately 127 plasmids per cells (0.2 µM) and approximately 5000 enzyme chimeras/cell (8 µM) (Supplementary Figure S3a), we determined that the zinc fingers should have sub-µM affinity. "
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