Team:Slovenia/PROJECT/proof/program

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DNA program

What is a DNA program?

DNA program is a noncoding DNA sequence consisting of specific binding sites for DNA binding domains. These DNA binding domains can be linked to functional domains (e.g. enzymes). This is analogous to the recognition of RNA codons by anticodons on aminoacyl tRNA, where the aminoacyl tRNA corresponds to the DNA binding domain with functional protein domain. When those functional domains assemble on a DNA program they are brought closer together in a defined order. By changing the places of binding sites on a DNA program it is possible to change the sequence of events (e.g. course of the reaction in case of the enzymes).

Figure 1: Analogy between central biological dogma and our idea of coding beyond triplets is preseneted

Selection and importance of spacer sites

Binding sites for three-fingered zinc fingers span 9 nucleotides but can be extended to 18 base pair recognition motifs for longer zinc fingers, spanning from one to two DNA duplex helical turns, respectively. Binding sites for DNA-binding proteins are separated by spacers, which are nucleotides that are not occupied by DNA-binding proteins. The length of the spacer sequence is not coincidental. The selection follows three dimensional structure of a DNA molecule. One turn of DNA helix is 10,5 base pairs long, which roughly overlaps with the length of a DNA molecule encircled by one zinc finger domain recognising and binding to 9 base pairs. In order to have functional units on the same side of a DNA molecule serving as a DNA program, it is of high importance to select the right spacer length. This is the case when having split functional units attached to DNA binding domains as well as when biosynthetic enzymes are selected for functional domains. Double helix of DNA defines on which side of the helix the functional domain will be attached, which is defined by the length of the spacer between DNA domain binding sites: spacer of 1 or 2 nucleotides positions them very close, while the spacer of five nucleotides positions the neighboring two functional domains to the opposite sides of the helix.

Figure 2: Role of the spacer length between binding sites in a DNA program.

We employed an in-house computer software in order to predict appropriate spacer sites, which selects the base pairs in such a way that overlapping of recognition motifs wouldn't occur and disturb the sequential binding of selected synthetic zinc fingers. Two base pair spacer sequence was selected by default based on the literature since it has been shown that it leads to efficient split GFP reassembly. Increased length of a spacer would be particularly useful for the assembly of large protein functional domains that exceed 3.5 nm, which is the pitch of B-type DNA duplex.

Variability of DNA program

Another quality of the idea of DNA program is it's variability. If we take only the most characterized DNA binding domains that bind 9 base pair motifs, they can in theory form 262.144 unique combinations. This is over 4000-fold increase over 64 possibilities within a DNA triplet code. Furthermore, DNA binding domains have been characterised that bind to 18 base pairs which increases the possibilities even further. The main advantage of our approach is that the sequence of DNA binding motifs are basically not constrained, we can select a DNA sequence for which we have the well characterized DNA binding protein available.

Applications of DNA programs and beyond

The most promising direction of the application of DNA programs probably lies in biosynthetic pathways. The approach can be applied to many other biosynthetic pathways where enhanced production and/or intermediate substrate channelling is desired to avoid unwanted metabolite flows. A possibility of implementing simple information processing circuits such as DNA based logical gates using split/FRET system can also be envisaged but was not further investigated during the project.

Cloning strategy to increase DNA program copy number

For the increased production of biosynthetic products multiple copies of DNA program should be beneficial. We employed cloning of multiple copies of a DNA program simultaneously. This was achieved by ordering overlapping 5' phosphorylated DNA program primers. When annealed they were ligated, blunt-ended with T4 polymerase and cloned into the vector. A scheme of the cloning strategy is shown below:

Figure 3: Cloning strategy to increase DNA program copy number.

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-<div id="naslov">proof of principle</div>
+<div id="naslov">DNA program</div>
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 <!-- OD TU NAPREJ PIŠI BESEDILO -->
-<p>&nbsp;</p>
+__TOC__
-The main idea of our project was to use DNA-binding domains, e.g. zinc fingers specific for unique target elements within a predesigned DNA program sequence, and show their ability to act as anchors for various protein functional domains (e.g. enzymes and split fluorescent proteins). Our main goal was to first characterize binding properties of DNA binding factors and demonstrate that their binding could be defined by the order of DNA target elements on DNA program sequence and later use them in an application, such as biosynthesis.
-<p>&nbsp;</p>
+<h2>What is a DNA program?</h2>
-In order to prove the concept described above we had to:
-<p style="margin-left: 48px;">1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; select the appropriate DNA-binding domains,</p>
+DNA program is a noncoding DNA sequence consisting of specific binding  sites for DNA binding domains. These DNA binding domains can be linked  to functional domains (e.g. enzymes). This is analogous to the recognition of RNA codons by anticodons on aminoacyl tRNA, where the aminoacyl tRNA corresponds to the DNA binding domain with functional protein domain. When those functional domains assemble on a DNA program they are brought  closer together in a defined order. By changing the places of  binding sites on a DNA program it is possible to change the sequence of  events (e.g. course of the reaction in case of the enzymes). <br>
-<p style="margin-left: 48px;">2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; demonstrate their specific binding to DNA motifs,</p>
-<p style="margin-left: 48px;">3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; show that the addition of functional domain does not interfere with their binding to DNA,</p>
-<p style="margin-left: 48px;">4.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; show that we can bind several adjacent DNA-binding proteins linked to functional protein &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;domains to the DNA program and demonstrate the function of such an assembled protein &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;complex.</p>
+[[Image:Slo tRNA gor.png|thumb|center|550px|'''Figure 1:''' Analogy between central biological dogma and our idea of coding beyond triplets is preseneted]]
-<p>&nbsp;</p>
-There are many proteins domains that bind to defined DNA sequences, such as restriction endonucleases, transcription factors, zinc fingers, transcriptional activator like (TAL) elements, etc.
-<p>&nbsp;</p>
+<h2>Selection and importance of spacer sites</h2>
-We found zinc finger domains most appropriate for our purpose, since they can be designed to bind selected DNA sequence through concatenation of small domains called fingers that recognize DNA trimers. There are zinc fingers that recognize from 9 up to 18 nucleotides. Recently the molecular code of DNA recognition by TAL effectors was deciphered (Boch et al., 2009), which provides another tool to design sequence specific DNA recognition proteins.
-<p>&nbsp;</p>
+Binding sites for three-fingered zinc fingers span 9 nucleotides but can  be extended to 18  base pair recognition motifs for longer zinc fingers, spanning from one  to two DNA duplex  helical turns, respectively. Binding sites for DNA-binding proteins are separated by spacers, which are nucleotides that are not occupied by DNA-binding proteins. The length of the spacer sequence is not coincidental. The selection follows three dimensional structure of a DNA molecule. One turn of DNA helix is 10,5 base pairs long, which roughly overlaps with the length of a DNA molecule encircled by one zinc finger domain recognising and binding to 9 base pairs. In order to have functional units on the same side of a DNA molecule serving as a DNA program, it is of high importance to select the right spacer length. This is the case when having split functional units attached to DNA binding domains as well as when biosynthetic enzymes are selected for functional domains. Double helix of DNA defines on which side of the helix the functional domain will be attached, which is defined by the length of&nbsp; the spacer between DNA domain binding sites: spacer of 1 or 2 nucleotides positions them very close, while the spacer of five nucleotides positions the neighboring two functional domains to the opposite sides of the helix.<br>
-Two zinc fingers were already present in the Registry: Gli1 and ZNF HIVC, however their functionality has not been shown. In order to test our idea additional zinc fingers needed to be obtained (see list and characterization of parts - [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2010&amp;group=Slovenia link]).
-<p>&nbsp;</p>
+[[Image:slo_dna_scafold2.jpg|thumb|center|700px|'''Figure 2:''' Role of the spacer length between binding sites in a DNA program.]]
-We selected zinc finger proteins Tyr456, Zif268, PBSII, Blues and&nbsp;Jazz and order synthetic genes from GeneArt company. The first aim of our project was to show binding of&nbsp;zinc finger proteins to a specific DNA target sequence.
-<p>&nbsp;</p>
+We employed an in-house computer software in order to predict appropriate spacer sites, which selects the base pairs in such a way that overlapping of recognition motifs wouldn't occur and disturb the sequential binding of selected synthetic zinc fingers. Two base pair spacer sequence was selected by default based on the literature since it has been shown that it leads to efficient split GFP reassembly. Increased length of a spacer would be particularly useful for the assembly of large protein functional domains that exceed 3.5 nm, which is the pitch of B-type DNA duplex.<br><br>
-We tested binding of zinc fingers with added protein domains to DNA using several techniques: electrophoretic mobility shift analysis (EMSA), surface plasmon resonance (SPR), our new universal detection device composed of following two parts: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K323088 BBa_323088] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K323089 BBa_323089] for qualitative and quantitative analysis of DNA binding properties of selected DNA binding domains <em>in vivo</em> based on beta-galactosidase reporter.
-<p>&nbsp;</p>
+<h2>Variability of DNA program</h2>
-The first step providing proof of the principle was to test whether zinc fingers assemble on a DNA scaffold with the cognate binding sites&nbsp;using split fluorescent protein reconstitution. Fluorescent proteins were split into two overlapping segments and fused to different zinc fingers that should bind to adjacent sites on DNA program.
-<p>&nbsp;</p>
+Another quality of the idea of DNA program is it's variability. If we take only the most characterized DNA binding domains that bind 9 base pair motifs, they can in theory form 262.144 unique combinations. This is over 4000-fold increase over 64 possibilities within a DNA triplet code. Furthermore, DNA binding domains have been characterised that bind to 18 base pairs which increases the possibilities even further. The main advantage of our approach is that the sequence of DNA binding motifs are basically not constrained, we can select a DNA sequence for which we have the well characterized DNA binding protein available. <br><br>
-Once the reconstitution of pairs of fluorescent proteins was confirmed we could test binding of four zinc fingers next to each other, which led to the F&ouml;rster resonance energy transfer (FRET). These experiments were performed in mammalian cells, since emmitting fluorescence in mammalian cells is relatively easy to detect under a confocal microscope.
-<p>&nbsp;</p>
+<h2>Applications of DNA programs and beyond</h2>
-<p>&nbsp;</p>
-[[Image:SLOFret.png|frame|'''Schematic representation of the determination of the assembly of split fluorescent proteins along DNA in order to achieve the FRET effect.''']]
+The most promising direction of the application of DNA programs probably lies in biosynthetic pathways. The approach can be applied to many other biosynthetic pathways where enhanced production and/or intermediate substrate channelling is desired to avoid unwanted metabolite flows. A possibility of implementing simple information processing circuits such as DNA based logical gates using split/FRET system can also be envisaged but was not further investigated during the project.<br><br>
-<p>&nbsp;</p>
-Results on the following pages show that we characterized binding properties of 6 zinc fingers to their respective DNA elements, including two (Gli1 and HIVC) that have previously been deposited in the Registry, but not characterized. We demonstrated proof of the principle of DNA-guided assembly of four different zinc finger fusion proteins by&nbsp;FRET. FRET&nbsp;device consists of five elements: four fusion proteins with different DNA binding domains and a separate DNA program sequence. It has a potential to be used for the purpose of information processing and pattern recognition, however, other fused functional protein domains may be more versatile in comparison to the fluorescent proteins and FRET.
+<h2>Cloning strategy to increase DNA program copy number</h2>
-<p>&nbsp;</p>
-<p>&nbsp;</p>
+For the increased production of biosynthetic products multiple copies of DNA program should be beneficial. We employed cloning of multiple copies of a DNA program simultaneously. This was achieved by ordering overlapping 5' phosphorylated DNA program primers. When annealed they were ligated, blunt-ended with T4 polymerase and cloned into the vector. A scheme of the cloning strategy is shown below:<br>
+[[Image:slo_program.png|thumb|center|700px|'''Figure 3:''' Cloning strategy to increase DNA program copy number.]]
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