Biomod/2011/Slovenia/BioNanoWizards/ideaproteinaddons

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Protein add-ons



Protein functionalization of DNA has so far utilized a limited number of orthogonal approaches, using nucleic acid-protein conjugates (Wilner, 2009), aptamer-protein interactions (Rinker, 2008), antibody-antigen interactions (He, 2006; Williams, 2007) and streptavidin-biotin system (Yan, 2003; Kuzuya, 2009; Voight, 2010). Sacca and co-workers demonstrated the use of three different protein tags at the same time: biotin-streptavidin interaction and two suicide ligands for their specific enzymes (Sacca, 2010). These systems are based mainly on incorporation of specifically labeled oligonucleotides.

We proposed a more general approach for position-specific protein binding to the surface of DNA origami, which has to comply with several criteria:

  • strong binding affinities (Kd preferably in the nanomolar or subnanomolar range),
  • simultaneous binding of several molecules using the same approach and reaction conditions,
  • technological feasibility in terms of cost and technology used.


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Figure 2: Schematic

representation of six-finger ZFP attached to its DNA target. Each zinc finger motif

composed of ββα fold binds specifically to 3 bp of DNA.

Last year <a 

href="http://2010.igem.org/Team:Slovenia">Team Slovenia at

iGEM2010</a> competition proposed a scaffold-assisted biosynthetic pathway utilizing linear dsDNA as a program and zinc finger proteins as binding domains. This approach increased production of trans-resveratrol in bacteria 5-fold in the presence of DNA program which arranged the enzymes in the correct order (Conrado, 2011).

Zinc finger proteins (ZFPs) are the most widespread and best characterized DNA-binding protein domains to date. These small structural motifs form coordination bonds with zinc cations to stabilize their ββα fold. Each zinc finger recognizes and binds three consecutive base pairs of a double stranded DNA in a sequence-dependent manner (Figure 2). Their specificity is based on interactions between amino acid side chains of the zinc finger α-helix and the DNA.

Moreover, zinc finger proteins can be designed by direct protein fusion of several successive zinc fingers which gives us access to an almost unlimited pool of proteins (at least 700 well characterized in the ZiFDB database), which specifically and predictably target distinct DNA sequences. Binding affinity of such zinc finger proteins to their target DNA sequences depends mainly on the number of successive zinc fingers used. Therefore, six finger proteins had been reported to have subnanomolar and even picomolar binding affinities.


A DNA origami add-on approach proposed by BioNanoWizards

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Figure 3: Position

specific functionalization of DNA origami with selected protein functions. Step 1: Desired functional protein domains are combined with any of the characterized 700 ZFPs into chimeric proteins. Step 2: Staples at selected positions on DNA origami are replaced with staples with added hairpin that incorporates the recognition sequence of the ZFPs selected in step 1, DNA origami with modified staples is annealed. Step 3: ZFP-functional domain chimeras are added to DNA origami and bind to the

selected positions.

Full potential of zinc fingers could be realized by fusion to selected functional protein domains, e.g. nucleases, recombinases and others, which have already been used to engineer genomes. In such systems zinc finger proteins serve for targeting functional domains to specific sites on DNA. This idea set the foundation for protein DNA origami add-ons. By inserting DNA hairpins into staple strands at specific positions on DNA origami, one can easily direct binding of such functional domains through zinc finger protein-target DNA interactions. Such an approach offers immediate high-tech applications such as <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/applabonchip">lab-on-a-nanochip</a>,

<a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosensors">nanobiosensors</a>,

sequential biosynthetic route setups or <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosynthteticcompartments">construction

of biosynthetic organelles</a>.

DNA binding proteins might be also used for structural purposes, e.g. to combine several DNA nanostructures via protein tethers, containing ZFPs, which is described in the next section.


  • Conrado RJ, Wu GC, Boock JT, Xu H, Chen SY, Lebar T, Turnšek J, Tomšič N, Avbelj M, Gaber R, Koprivnjak T, Mori J, Glavnik V, Vovk I, Benčina M, Hodnik V, Anderluh G, Dueber JE, Jerala R, DeLisa MP (2011) DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic Acids Res. in press
  • He Y, Tian Y, Ribbe AE, Mao C (2006) Antibody Nanoarrays with a pitch of 20 nanometers J. Am. Chem. Soc. 128: 12664-12665.
  • Kuzuya A, Kimura M, Numajiri N, Koshi N, Ohnishi T, Okada F, Komiyama M (2009) Precisely programmed and robust 2D streptavidin nanoarrays by using periodical nanometer-scale wells embedded in DNA origami assembly ChemBioChem 10: 1811-1815.
  • Rinker S, Ke Y, Liu Y, Chhabra R (2008) Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding Nat. Nanotech. 3: 418-422.
  • Sacca B, Meyer R, Erkelenz M, Kiko K, Arndt A, Schroeder H, Rabe KS, Niemeyer CM (2010) Orthogonal protein decoration of DNA origami. Angew. Chem. 49: 9378-83.
  • Voight NV, Torring T, Rotaru A, Jacobsen MF, Ravnsbaek JB, Subramani R, Mamdouh W, Kjems J, Mokhir A, Besenbacher F, Gothelf KV (2010) Single molecule chemical reactions on DNA origami Nat. Nanotechnol. 5:200-203.
  • Williams BAR, Lund K, Liu Y, Yan H, Chaput JC (2007) Self-assembled peptide nanoarrays: an approach to studying protein-protein interactions Angew. Chem. 119: 3111-3114.
  • Wilner OI, Weizmann Y, Gill R, Lioubashevski O, Freeman R, Willner I (2009) Enzyme cascades activated on topologically programmed DNA scaffolds Nat. Nanotech. 4: 249-254.
  • Yan H, Park SH, Finkelstein G, Reif JH, LaBean TH (2003) Design and construction of double-decker tile as a route to three-dimensional periodic assembly of DNA Science 301: 1882-1884.

 

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