Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method.
The C2H2 zinc finger is the most commonly utilized framework for engineering DNA-binding domains with novel specificities. Many different selection strategies have been developed to identify individual fingers that possess a particular DNA-binding specificity from a randomized library. In these experiments, each finger is selected in the context of a constant finger framework that ensures the identification of clones with a desired specificity by properly positioning the randomized finger on the DNA template. Following a successful selection, multiple zinc-finger clones are typically recovered that share similarities in the sequences of their DNA-recognition helices. In principle, each of the clones isolated from a selection is a candidate for assembly into a larger multi-finger protein, but to date a high-throughput method for identifying the most specific candidates for incorporation into a final multi-finger protein has not been available. Here we describe the development of a specificity profiling system that facilitates rapid and inexpensive characterization of engineered zinc-finger modules. Moreover, we demonstrate that specificity data collected using this system can be employed to rationally design zinc fingers with improved DNA-binding specificities.
Meng
Xiangdong
X
Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation St, Worcester, MA 01605, USA.
Thibodeau-Beganny
Stacey
S
Jiang
Tao
T
Joung
J Keith
JK
Wolfe
Scot A
SA
D001419:Bacteria / Q000235:genetics; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D015202:Protein Engineering / Q000379:methods*; D014157:Transcription Factors / Q000737:chemistry; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA