Validated zinc finger protein designs for all 16 GNN DNA triplet targets.

The Cys(2)-His(2)-type zinc finger DNA-binding proteins can be engineered to bind specifically to many different DNA sequences. A single zinc finger typically binds to a 3-4-base pair DNA subsite. One strategy for design is to identify highly specific fingers that recognize each of the 64 possible DNA triplets. We started with a subgroup of the 64 triplets, the GNN-binding fingers. The GNN-binding fingers have been examined in several studies, but previous studies did not produce specific fingers for all of the 16 GNN triplets. These previous studies did not provide any information on the possible positional or context effects on the performance of these fingers. To identify the most specific design and take the possible positional effects into consideration, we did a large-scale site selection experiment on our GNN designs. From this study, we identified very specific fingers for 14 of the 16 GNN triplets, demonstrating for the first time a clear positional dependence for many of the designs. Further systematic specificity study reveals that the in vivo functionality of these zinc finger proteins in a reporter assay depends on their binding affinities to their target sequences, thus giving a better understanding of how these zinc finger proteins might function inside cells.     

C2H2型锌指蛋白结合的DNA序列预测方法的研究进展

C2H2型锌指蛋白是哺乳动物中数量最多的一类转录调控因子.C2H2型锌指蛋白中含有的C2H2型锌指基序多是不相同的,表明它们很可能结合不同的DNA序列,从而调控不同的基因,行使多样化的调控功能.然而,目前大多数C2H2型锌指蛋白结合的DNA序列仍不明确,这阻碍了C2H2型锌指蛋白的功能研究.目前,针对C2H2型锌指蛋白的靶序列预测已有一些初步的研究.本文介绍了C2H2型锌指基序与DNA结合的经典模式,并对C2H2型锌指蛋白靶序列预测方法中所用到的算法、训练集、金标准数据集及相应工具进行了全面系统的总结归纳,旨在丰富对C2H2型锌指蛋白靶序列预测原理和工具的认识,为C2H2型锌指蛋白靶序列的精确预测和更深入的功能研究打下基础.     

Exploiting the recognition code for elucidating the mechanism of zinc finger protein-DNA interactions

Background

Engineering zinc finger protein motifs for specific binding to double-stranded DNA is critical for targeted genome editing. Most existing tools for predicting DNA-binding specificity in zinc fingers are trained on data obtained from naturally occurring proteins, thereby skewing the predictions. Moreover, these mostly neglect the cooperativity exhibited by zinc fingers.

Methods

Here, we present an ab-initio method that is based on mutation of the key α-helical residues of individual fingers of the parent template for Zif-268 and its consensus sequence (PDB ID: 1AAY). In an attempt to elucidate the mechanism of zinc finger protein-DNA interactions, we evaluated and compared three approaches, differing in the amino acid mutations introduced in the Zif-268 parent template, and the mode of binding they try to mimic, i.e., modular and synergistic mode of binding.

Results

Comparative evaluation of the three strategies reveals that the synergistic mode of binding appears to mimic the ideal mechanism of DNA-zinc finger protein binding. Analysis of the predictions made by all three strategies indicate strong dependence of zinc finger binding specificity on the amino acid propensity and the position of a 3-bp DNA sub-site in the target DNA sequence. Moreover, the binding affinity of the individual zinc fingers was found to increase in the order Finger 1 < Finger 2 < Finger 3, thus confirming the cooperative effect.

Conclusions

Our analysis offers novel insights into the prediction of ZFPs for target DNA sequences and the approaches have been made available as an easy to use web server at http://web.iitd.ac.in/~sundar/zifpredict_ihbe

     

Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA

Cys2-His2 zinc fingers are one of the most common types of DNA-binding domains. Modifications to zinc-finger binding specificity have recently enabled custom DNA-binding proteins to be designed to a wide array of target sequences. We present here a 1.96 A structure of Aart, a designed six-zinc finger protein, bound to a consensus DNA target site. This is the first structure of a designed protein with six fingers, and was intended to provide insights into the unusual affinity and specificity characteristics of this protein. Most protein-DNA contacts were found to be consistent with expectations, while others were unanticipated or insufficient to explain specificity. Several were unexpectedly mediated by glycerol, water molecules or amino acid-base stacking interactions. These results challenge some conventional concepts of recognition, particularly the finding that triplets containing 5'A, C, or T are typically not specified by direct interaction with the amino acid in position 6 of the recognition helix.     

Rational design of DNA sequence-specific zinc fingers

We developed a rational scheme for designing DNA binding proteins. The scheme was applied for a zinc finger protein and the designed sequences were experimentally characterized with high DNA sequence specificity. Starting with the backbone of a known finger structure, we initially calculated amino acid sequences compatible with the expected structure and the secondary structures of the designed fingers were then experimentally confirmed. The DNA-binding function was added to the designed finger by reconsidering a section of the amino acid sequence and computationally selecting amino acids to have the lowest protein-DNA interaction energy for the target DNA sequences. Among the designed proteins, one had a gap between the lowest and second lowest protein-DNA interaction energies that was sufficient to give DNA sequence-specificity.