Zinc Finger Proteins (ZFPs)
Our proprietary technology is based upon the engineering of a naturally occurring class of DNA transcription factors called zinc finger DNA-binding proteins (ZFPs). Naturally occurring ZFP proteins are highly conserved across species and are used by organisms from yeast to man to control gene expression. They have two domains, the first is the ZFP portion or recognition domain, which recognizes and specifically binds to a particular DNA sequence. The second component is a functional domain which when brought into close proximity with a gene has a biologic effect on that gene (see below).
Figure 1.: Schematic of the structure of Sangamo’s engineered ZFP TFs and ZFNs
The two-component structure of our engineered ZFPs is modeled on the structure of naturally occurring transcription factors. We can engineer ZFPs to bind to virtually any DNA sequence. We can combine the engineered ZFPs with a variety of different functional domains to generate novel ZFP transcription factors (ZFP TFs) which are proteins that can activate or repress gene expression. Additionally, we can create novel ZFP Nucleases (ZFNs) that enable us to specifically modify gene sequences in a variety of ways.
Figure 2: Schematic of the structure of Sangamo’s engineered ZFP transcription factors and nucleasesActing at the DNA level enables us to affect biological outcomes that cannot be achieved by acting at the protein or the RNA level. As the genetic material of all living organisms is DNA this means that our technology has broad applications across species.
Of the many different DNA binding motifs that have evolved over time, C2 H2 zinc finger DNA binding proteins have proven to be the most versatile and nature has fully exploited their useful properties.
Figure 3: Genome-wide comparison of transcriptional activator families in eukaryotes. This histogram shows that C2H2 zinc finger class of DNA binding proteins is the most abundant class of transcription factor in all of the species that they authors surveyed. Tupler R, Perini G, Green MR (2001). Nature 409: 832-833.
C2H2 zinc fingers are found in 2% of all human genes, and they are by far the most abundant class of DNA-binding domains found in human transcription factors. Their structure makes them an ideal framework for engineering to bind to selected target sequences. This is because of their compact modular structure and the fact that they can be stitched together to bind to longer DNA sequences in a predictable way. Each “module” or finger recognizes and binds to 3-4 base pairs of DNA. Three such fingers can be joined together to bind a 9-base pair sequence and correspondingly 6 fingers an 18 base pair sequence. From the structural analysis of such proteins the actual amino acids that make contact with the DNA have been identified and are located at the same position in each finger. Varying these amino acids enables the ZFP to bind to different DNA sequences.