General introduction
The goal of providing an universal restriction enzyme was approached with two design strategies. The first strategy evolves around novel protein fusion constructs combining the cleavage domain of the Type IIs restriction enzyme FokI with hapten binding anticalins, which are then guided to their target sites by a modified oligonucleotide. The second approach aimed at converting the Argonaute proteins from thermophilic organisms from an RNase to an DNase activity while accepting a DNA guide oligonucleotide. For the Fok-based strategy, several variations were tested. In both projects important milestones were reached. Most importantly, we demonstrated guided cleavage by one of our Fok-based fusion constructs. In the following we introduce these two projects and then list the highlights of our work with links to detailed descriptions of each project part.
Both strategies rely on locating the universal restriction enzyme at the cleavage site with adapter or guide oligonucleotides. This is in contrast to previous designs which either use chemical linkage of an oligonucleotide to a nuclease or genetic fusion with a DNA binding domain. Previous fusion protein approaches have the conceptual disadvantage that for each target sequence a special protein has to be designed, expressed, purified, and stored. We only need to produce one protein. The oligonucleotide planning is aided by readily available PCR primer design programs and there are virtually no limits to define the cleavage site. Modified oligonucleotides can be ordered from many suppliers at low cost with short delivery times and easily can be stored long term. In in-vitro applications hybridization of the oligonucleotide is easily achieved also to double strand by heating and cooling as it is well known from PCR procedures. In case of the Argonaute proteins from thermophiles we can assume that they survive several cylces of heat dissociation and annealing. In case of the Fok we plan to introduce thermostability by directed evolution. In addition, other groups are working with oligonucleotides forming triple helices and their general hybridization with any sequence or with peptide nucleotide acids which provide higher stability and sneak in existing double helices. These technologies are compatible with our approach.
In both of our universal restriction enzyme strategies we do not cut the double strand but rather nick the stand opposite to our guide oligonucleotide. Thus, our guide oligonucleotide only needs to be added only in catalytic quantities. The nicking feature is already present in the Argonaute proteins. In the case of the universal Fok-based enzyme we use a heterodimer design combined with cleavage inactivating mutations for one monomer.
Adapter-guided DNA cleavage: Fok-Anticalin fusions
The restriction endonuclease FokI from Flavobacterium okeanokoites is a well studied protein. It consists of two domains, a DNA recognition domain and a DNA cleavage domain. Upon recognition of one target site and dimerization it cleaves the DNA nine bases apart from the recognition site. Several groups reported experiments to uncouple the cleavage and restriction domains of FokI and created a novel site-specific endonucleases by linking the cleavage domain to zinc finger proteins (Miller et al. 2007).
For our project we combined two previous research results and generated a Fok cleavage heterodimer comprising an enzymatically active and inactive monomer. For the catalytic active Fok partner, named Fok_a, as well as for the catalytic inactive Fok partner, Fok_i, the association interface was mutated to disfavor homodimerization and promote heterodimerization. In Fok_i amino acid exchanges led to the inactivation.
The two heterodimeric partners were fused to anticalins binding different adapter molecules. Fok_a is genetically fused to a digoxigenin-binding anticalin (DigA) and Fok_i to a fluorescein-binding anticalin (FluA). The adapter molecules digoxigenin and fluorescein are common modifications linked to oligonucleotides thus mediating the binding to the DNA site of interest. Two modifications allow for a better spatial control of the cleavage site. On the target site Fok_i and Fok_a constructs are brought into close proximity and can form a heterodimer. The inactive Fok domain will serve as an activator of the active Fok domain, wich cuts one strand of the DNA. Our structural ‘3D’ models, indicate that Fok domains can be positioned in such a way that Fok_a will cleave the target DNA, and Fok_i would be directed towards the modified oligonucleotide. Different linkers were designed and fused between cleavage domain and the anticalin binding moieties to test for the optimal distance. In addition we generated a monomeric Fok fusion construct, to enable phage display and te4st a further setting requiring onlyone bionding domain and hapten. Furthermore, we made a Fok cleavage domain fusion with a coiled coil based DNA binding domain, because we can combine these with existing light switchable inhibitors, which prevent DNA binding. As a result we will obtaina light switchable restriction enzyme.
Towards a universal restriction enzyme based on Argonaute (Ago) proteins
Ago proteins origin; phage display;
Milestones
To reach our goal within the short given time frame we started several subprojects in parallel. Our subprojects listed here are defined along these projects.
Designing of the constructs was aided by extensive model building and analysis of the spatial orientation of the different proteins and oligonucleotides used. All designed and constructed parts feature full BioBrick compatibility and in addition allow for the construction of fusion proteins based on the RFC 25 (Freiburg) cloning standard. Cloning of the respective parts was followed by expression purification and analysis. Despite previous literature data our active Fok construct was significantly toxi to cells when expressed and we tested periplasmic expression with, which exports the nascent polypeptide chain before folding. In our experiments we addressed the following questions:
• Structural Model bulding
• Design of protein fusion parts
• Cloning of anticalin Fok fusions
• Cloning of a monoeric Fok construct and of a Jun/Fos directed Fok construct
• Expression and purification of constructs
• In vitro assays
• In vivo asssays
• Phage Display of an Ago protein
• Modeling of assembly and cleavage with differential equations
• An international survey of laymen on synthetic biology
In short, we successfully addressed . Experiments to##### are ongoing.
For our modeling analyses we constructed various sets of differential equations describing our synthetic receptors and predicted split protein activation behaviour.
The labs of Kristian Müller and Katja Arndt provided all technology and support for the project.
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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.
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| complete universal restriction enzyme.
Blue: DNA strand; Red: two 16bp
long Oligos, tags as indicated in the
picture. Ochre: Fluorescein A
binding lipocalin; Orange: digoxigenin binding
lipocalin; Yellow:
tagged Base with C6 linkers and attached tags; light Blue: inactive
FokI cleavage domain; Red: FokI active
cleavage domain; Green: three
catalytically active aminoacids; White: two Calcium ions |
The place where all the
cutting events will take place in our scenery
is the thermocycler. Therefore all the ingredients, i.e. chromosomal or
plasmid DNA of interest, modified oligonucleotides and the different
heterodimers Fok_a and Fok_i are mixed together. At high temperatures
the DNA will denaturate allowing the different partners to find each
other, cut the DNA, and fall apart in course of one thermocycle. The
whole procedure can be repeated undefinately. To reach this step, we
need to improve the thermostability of our enzyme via phage display.
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 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).
Additionally, we provide
an alternative way of binding by the coupling
of the cleavage domain to the transcription factor Fos. This concept
also includes the activity regulation by photo-switching. We are also
modifying an argonaute protein into a DNA endonuclease using phage
display technics.
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| Scheme of 1. Cutting, 2. Binding, 3. Dissociation of the programmable restriction enzyme
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TFO or PNA respectively used to cut dsDNA
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Project Report
