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Project Report
We focused our project on coupling and optimizing the characteristics
of a restriction endonuclease with short oligonucleotides to develop a
programmable and highly specific enzyme-oligo-complex. As a restriction
endonuclease we chose the cleavage domain of the well studied
endonuclease FokI from Flavobacterium okeanokoites. Normally FokI acts
as a homodimer, each dimer divided in cleavage and restriction domain.
Chandrasegaran and Miller have already made experiments to uncouple the
cleavage and restriction domains of FokI and created a novel
site-specific endonuclease by linking the cleavage domain to zinc
finger proteins.
For our project we generated two Fok heterodimers (Miller, Nature biotech, 2007). For the catalytic active Fok partner, named Fok_a, the first 1158 nucleotides, i.e. the recognition domain, were deleted and glutamate 490 was switched to lysine (GAA->AAA) as well as isoleucine 538 to lysine (ATC->AAA) for the heterodimer formation. For the catalytic inactive Fok partner, named Fok_i, the heterodimeric amino acids glutamine 486 was switched to glutamate (CAA->GAA) and isoleucine 499 to leucine (ATC->CTG) and the catalytic amino acids aspartate 450 was switched to alanine (GAC->GCG) and aspartate 467 to alanine (GAT->GCG).
The two heterodimeric partners were fused to different anticalins binding different adapter molecules. Thus Fok_i is fused to anticalin on Fluorescein and Fok_a to anticalin on Digoxigenin. These adapter molecules are linked to oligonucleotides mediating the binding of the DNA site of interest. Now the heterodimerization comes into play. If the different Fok_i and Fok_a constructs bind their target oligos and come together, the inactive domain will serve simply as an activator of the active domain, cutting only one strand of the DNA. In our 3D models we showed that Fok domains are positioned in such a way that Fok_a will cut the DNA and Fok_i the modified oligonucleotide. Thus the inactivation of Fok_i allows the reuse of our oligonucleotides. Different linkers were designed and fused between cleavage domain and binding protein to test the optimal distance to preserve the most possible flexibility and most possible precision of the heterodimeric Foks.
For our project we generated two Fok heterodimers (Miller, Nature biotech, 2007). For the catalytic active Fok partner, named Fok_a, the first 1158 nucleotides, i.e. the recognition domain, were deleted and glutamate 490 was switched to lysine (GAA->AAA) as well as isoleucine 538 to lysine (ATC->AAA) for the heterodimer formation. For the catalytic inactive Fok partner, named Fok_i, the heterodimeric amino acids glutamine 486 was switched to glutamate (CAA->GAA) and isoleucine 499 to leucine (ATC->CTG) and the catalytic amino acids aspartate 450 was switched to alanine (GAC->GCG) and aspartate 467 to alanine (GAT->GCG).
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| Association of linker FluA and Dig with DNA and Fok_a and Fok_i monomers |
The two heterodimeric partners were fused to different anticalins binding different adapter molecules. Thus Fok_i is fused to anticalin on Fluorescein and Fok_a to anticalin on Digoxigenin. These adapter molecules are linked to oligonucleotides mediating the binding of the DNA site of interest. Now the heterodimerization comes into play. If the different Fok_i and Fok_a constructs bind their target oligos and come together, the inactive domain will serve simply as an activator of the active domain, cutting only one strand of the DNA. In our 3D models we showed that Fok domains are positioned in such a way that Fok_a will cut the DNA and Fok_i the modified oligonucleotide. Thus the inactivation of Fok_i allows the reuse of our oligonucleotides. Different linkers were designed and fused between cleavage domain and binding protein to test the optimal distance to preserve the most possible flexibility and most possible precision of the heterodimeric Foks.
