Rational design of DNA sequence‐specific zinc fingers

Abstract

Edited by Robert B. RussellKeywords: DNA-binding protein Protein design Specificity Affinity Energy gap a b s t r a c tWe 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.

Protein Design: Toward Functional Metalloenzymes

“…115–119 Multiple ZF mutations have been made to change how and where the domain will bind to and recognize DNA. 112,120–131 Much effort has focused on structural swapping, where the secondary structure is altered to combine either the native α -helix or β -sheet of a ZF and the opposite fold from another protein, but the binding site remains unaltered. 132–138 Last, many studies have been conducted on designing multidomain ZF proteins to bind to extended regions of DNA (>16 base pairs).…”

“…Much work has been conducted on structural studies of ZF proteins as well as studies that focus on the alteration of binding and recognition of the ZF to DNA. These types of studies will not be focus of this Review, but will be summarized with references here, and readers are encouraged to read the extensive reviews by Negi et al, Matthews et al, and Kaptein, for further direction to structural studies. ,, Structural and nucleic acid binding properties to elucidate DNA binding details are extensively outlined elsewhere. − Multiple ZF mutations have been made to change how and where the domain will bind to and recognize DNA. ,− Much effort has focused on structural swapping, where the secondary structure is altered to combine either the native α-helix or β-sheet of a ZF and the opposite fold from another protein, but the binding site remains unaltered. − Last, many studies have been conducted on designing multidomain ZF proteins to bind to extended regions of DNA (>16 base pairs). − Linker-domains, where two sections of ZF regions are separated by a linker, were also introduced for ZFs to bind to an extended region of DNA. − Alterations in DNA binding have also been used to result in bending the DNA upon binding, , and even protein stability studies to promote protein folding in the absence of the metal ions. − Further, Seelig and Szostak reported a novel RNA ligase designed by attaching a Zn­(Cys) 4 ZF protein with randomly mutated loop region onto an mRNA and using mRNA display to select for the optimal mutation . Besides DNA binding and transcription regulation, there are other biophysical parameters to be studied by exploiting the stability and the small size of ZF proteins.…”

DNA-binding One Zinc Finger (DOF) transcription factors in Vigna species: A review

“…Transcription factors can either make homodimers, heterdimers or both homo and heterodimers at the same time. This domain can eventually alter the function of the rest of the domains depending on its dimerization capability (Kono et al, 2012;Gupta et al, 2015). The cisregulatory site in the DNA binds to the particular amino acid (AA) sequences present in the DNA-binding site of the transcription factor that determines the affinity, specificity and selectivity of the transcription factors for the DNA.…”

“…The cisregulatory site in the DNA binds to the particular amino acid (AA) sequences present in the DNA-binding site of the transcription factor that determines the affinity, specificity and selectivity of the transcription factors for the DNA. This amino acid arrangement can affect the interaction between the TF and the DNA (Kono et al, 2012).…”

Balance between DNA‐binding affinity and specificity enables selective recognition of longer target sequences in vivo

“…This has enabled these enzymes to be applied to a variety of eukaryotic cells from Drosophila to mice, and several clinical tests in humans are proceeding . However, application of zinc‐finger nucleases has been limited because recognition is limited mainly to GC‐rich sequences . TALEN and CRISPR/Cas9, which were developed after zinc‐finger nucleases, can recognize a wider array of DNA sequences because they can choose nearly any combination of four nucleotides as target sites; the only exceptions are short conserved regions.…”