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 ZFPs are highly conserved across species and used by a wide range of organisms, from yeast to humans, to control gene expression. They have two domains, a ZFP portion or recognition domain, which recognizes and specifically binds to a particular DNA sequence, and a functional domain which, when brought into close proximity with a gene, has a biologic effect on the gene of interest (see below).
Figure 1. Schematic of the structure of Sangamo’s engineered ZFPs.
Sangamo's Engineered ZFPs
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 with singular specificity. 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 TFs and ZFNs. Acting at the DNA level enables us to effect 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 enables the broad application of our technology 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 the C2H2 zinc finger class of DNA binding proteins is the most abundant class of transcription factor in all of the species the 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 motifs found in human transcription factors. Their structure makes them an ideal framework to engineer singularly specific DNA target sequence binding. 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 and consistent 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 to 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.