Team:Freiburg bioware/Project

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FREiGEM



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.


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.








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.
Western Blot: His-Flu_a-Split_Fok_i in pEx; lanes: NEB prestained marker broad range, pool of elution fractions 2-5, empty lane, 3 positive controls Western Blot: His-Flu_a-Split_Fok_i in pEx; lanes: NEB prestained marker broad range, pool of elution fractions 2-5, empty lane, 3 positive controls
Scheme of 1. Cutting, 2. Binding, 3. Dissociation of the programmable restriction enzyme TFO or PNA respectively used to cut dsDNA




Modeling

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3D-Modeling

Structural modeling was an initial step towards our planned universal restriction enzyme. Using molecular display software we arranged published crystal structures of FokI, anticalins and DNA. The arrangement was guided by superimposition with further structures of enzyme bound DNA. The modeling defined spatial requirements for linker lengths and positioning of modifications within oligonucleotides.
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In vitro assays

After the cloning, expression and the purification of the Fok constructs we conducted several assays to analyse the activity of the enzyme. To establish the assay and as a reference for activity we used wildtype FokI. Binding of the modified nucleotides and enzymatic activity were tested with the Fok_i / Fok_a construct.
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Fok Monomer


In order to create a universal restriction enzyme we followed several ideas and models. The first and most promising idea we had was to create a Fok Monomer as it represents the optimal and easiest model for a universal restriction enzyme

If we are able to employ a monomeric enzyme, this protein would have a couple of advantages: Most importantly, we would no longer need two separate oligonucleotides to achieve specific binding and cleavage of the target DNA. Also, only one protein has to be purified – thus saving time and money. A scientific advantage is the option to optimize the monomer by phage display. Using this technology, we would have the chance to create a thermostable, specific, universal restriction enzyme whose DNA binding activity is only created by a single oligonucleotide.

Our goal was first, to create a Fok-monomer that is able to cut DNA without a primary dimerization step, and second, to show that our heterodimeric interface design works properly. To reach this, we had to clone all the required parts one after another, resulting in a huge fusion protein.

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Protein Expression and Purification


In order to produce and study our different protein constructs they had to be expressed in bacteria.  After cloning the individual parts into the pMA vector the complete expression products were then transferred into the pEx vector (see pEx vector ) and transformed into competent E. coli expression strains via heat shock. We used two different strains for the protein expression: E. coli BL21 de3 (Novagen) and E. coli RV308 (Maurer et al., JMB 1980). Both strains are suited for high-level protein expression.

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In vivo Assays



In vivo use of programmable restriction enzymes can provide the opportunity of genome engineering. Here we develop and test strategies for the application of our programmable restriction endonuclease.
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AGO

The argonaute proteins represent one of our side projects in creating a universal programmable endonuclease.

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Alternative way of binding: Jun/Fos

We also thought of an alternative way of binding of the heterodimeric Fok to the DNA avoiding the necessity of labelled oligos and their binding proteins using the binding domain of a transcription factor as a new adapter. We focused on the binding domain of the activator protein-1 (AP-1) a crucial transcription factor implicated in numerous cancers. The protein is composed by a series of dimers. Nine homologues of the AP-1 leucine zipper region have been characterized, among them c-Fos, c-Jun and semirational library-designed winning peptides FosW and JunW. Via leucin zipper they interact among each other and with their basic region they bind DNA.
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Cloning strategy

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