Part:BBa_K243001

Protein domain (inactive) of the restriction endonuclease FokI

This part is used as the inactive domain of our universal restriction endonuclease. It cuts DNA, when it is fused to the active protein domain of our universal restriction endonuclease BBa_K243000 and linked with specific oligonucleotides hybridized to DNA.

Introduction

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 into 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) and this part acts as the active cutting domain of our universal endonuclease.

History:Sequence specific nuclease(1968)

The researchers H.O. Smith, K.W. Wilcox, and T.J. Kelley (Johns Hopkins University 1968) were the first persons who isolated and characterized the first restriction nuclease whose functioning depended on a specific DNA nucleotide sequence. This was a big breakthrough for the genetic engineering as it gave the scientists a tool for working with DNA. Now, over forty years later, over 3000 restriction enzymes have been studied in detail and more than 600 of these are available commercially and are routinely used for DNA modification and manipulation in laboratories.

The present idea

The idea for an universal endonuclease starts with the need for a restriction enzyme, which is programmable to cut DNA at specific chosen sites. We chose FokI because previous studies indicated that this endonuclease was modifiable for the process of cutting and binding DNA. We developed a plan how it could work and started the work in the wet lab. The plan for the cutting and binding determines a special order of events for the function of the universal endonuclease. The process for cutting begins when two heterodimeric partners are 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.

Usage and Biology

The usage of an universal endonuclease could change the daily routine of a scientist who is working with DNA because the question where to cut with which enzyme isn't needed anymore. He is free to choose the cutting sequence and can receive any part that he wanted. The only thing is that he has to plan where to cut and order the suiting specific oligos.

Applications

Creating a universal restriction enzyme provides not only the possibility to improve routine cloning but also to enhance therapeutic gene repair via triplex technology. Many genetic diseases and especially the ones arising from single nucleotide polymorphisms (SNPs) or monogenetic disease can be alleviated by the replacement of mutated genes using this method. To cut double stranded DNA, the oligonucleotides have to be replaced by triple helix forming oligos (TFO). They can bind double-stranded DNA in homopurin- or homopyrimidine-rich areas. But developments are also made to widen the possible interaction domains of the DNA and hence make the TFOs as programmable as our conventional oligonucleotides. In case of the human genome of 3×10^9 bp size, a highly specific artifical endonuclease would be necessary to address the mutated gene explicitly. The used TFOs therefore have to possess a minimum length of 16 bp to cut just once in the human genome (4^16 bp = 4.3*10^9 bp).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]