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Self-Organizing Bio-structures NB2-2009 L.Duroux Lecture 5: DNA Self-Assembly Applications The trends in nano-fabrication • The miniaturization, top-down „„sizeshrinking‟‟ – microelectronics technology – pushing down the limits of size and – compactness of components and devices • The nanofabrication and nanomanipulation bottom-up – molecular nanotechnology – of novel nanolevel materials and methods – (e.g., near-field scanning microscopies) to – electrical devices built on carbon nanotubes – optical devices like optical sieves (69). • The supramolecular self-organization approach – complexity through self-processing, – self-fabrication by controlled assembly & hierarchical growth – connected operational systems Remember Nucleic Acids (DNA) and their Self-Assembly properties D • An example of a reciprocal exchange: Two DNA helices are connected by sharing two DNA strands (Seeman, 2001) C A B Oligonucleotides Advantages of nucleic acids as nanomaterials • Size: Ø of 1nm for ssDNA and Ø 2nm for dsDNA • Chemical stability and robustness • Production costs for synthesis are low • Self-assembly properties DNA as scaffold for nano- architectures 1. Using ssDNA as template to self-assemble nanostructures A simple case of ssDNA-functionalized micro-beads Polymer brush -> steric repulsion • Specific and reversible aggregation of micro-beads grafted with oligonucleotides • The key to reversibility is preventing the particles from falling into their van der Waals well at close distances Valignat et al, 2005. PNAS 102(12): 4225-29 T= 23¤C T= 50¤C Interaction Energies of micro-beads • Trick is: create a Uminimum well outside UvdW well • Balancing finely Urep and Udna • Limiting the number of base-pair bonds between two cDNAs Lennard-Jones Potential • Potential function of: – Depth of potential well (E) – Distance at which potential is zero (s) • Term in power 12 describes repulsive forces Directed Assembly of micro-beads with optical tweezers • Beads are immobilized on array of discrete optical traps • Optical tweezers to move the traps closer to trigger DNA hybridization Effect of ssDNA length and rigidity • Micro-beads manipulated with optical tweezers • Two types of DNA hybrids: “flexi” and “rigid” Biancaniello et al, 2005. Phys Rev Lett. 94:058302 Binding Energies as function of rigidity of ssDNA • For identical Tm (43.7¤C), “rigid” spacer gives stronger U well Effect of ssDNA density on aggregate structuration 14000/sphere 3700/sphere • DNA density of 14000 molecules / sphere lead to unstructured aggregates • DNA density of 3700 3700/sphere T >> Tm molecules / sphere lead to self-assembled crystallites 2. DNA tiles: the ”building bricks” N. Seeman: the father of DNA nanotechnology • Any type of ss or dsDNA secondary structure can be exploited to create geometric shapes by self-assembly • Typically, junctions and sticky-ends are exploited for this purpose Branch molecules and branch migration Dyad Axis of seq. symmetry Homologous duplexes Reciprocal exchange Stable branch junction No Axis of seq. symmetry No complement sequence in corners Stem formation on inexact complementary strands Creation of stable motifs with DNA by reciprocal exchange Combinatorial self-assembly of DNA nanostructures AFM pictures of DNA tiles combinations Topology measurements by AFM Motif formed by quadruple cross-over (QX) & Lattice A B The concept of DNA tiles Example with triangle motifs B Central core strands A C Side strands Horseshoe strands Lattices from SA of triangle motifs Brun et al, 2006 Creation of 3D tiles with QX motifs A B C 3D structures from DNA self-assembly (Seeman, 2003) A cube A truncated octahedron Another tiling process using tecto-squares Chworos et al., Science306, 2068 (2004). Applications of DNA lattices • Molecular Electronics: – Layout of molecular electronic circuit components on DNA tiling arrays. • DNA Chips: – ultra compact annealing arrays. • X-ray Crystallography: – Capture proteins in regular 3D DNA arrays. • Molecular Robotics: – Manipulation of molecules using molecular motor devices arranged on DNA tiling arrays. DNA as template for electrical nano-wires A step toward “nano-electronics” DNA for Molecular Lithography: principle Gazit, 2007. FEBS J. 274:317-322 DNA lithography: towards nanoelectronics Niemeyer, 2002. Science, 297:62-63. Conducting DNA-nanowires 4x4 DNA tile Yan et al, 2003. Science 301:1882-84 DNA-Templated Self-Assembly of Metallic Nanocomponent Arrays on a Surface DNA-Templated Self-Assembly of Metallic Nanocomponent Arrays on a Surface DNA-Templated Self-Assembly of Metallic Nanocomponent Arrays on a Surface Templated array of proteins on 4x4 nanogrids Biotinylated DNA 4x4 tiles Streptavidin • In nano-electronics designs: possibility to self-assemble proteins on DNA grid Nano-electronics components Metallization and conductivity measurements of DNA 4x4 tile ribbons 500 nm 500 nm Programmable Self-Assembly of DNA Computation by Self-assembly of DNA Tilings • Tiling Self-assembly can: – Provide arbitrarily complex assemblies using only a small number of component tiles. – Execute computation, using tiles that specify individual steps of the computation. • Computation by DNA tiling lattices: – Fist proposed by Winfree (1998) – First experimentally demonstrated by Mao, et al (2000) and N.C. Seeman (2000). Molecular-scale pattern for RAM-memory 3 components for DNA computing • DNA computing (Adleman, 1994) • Theory of tilings (Grunbaum and Sheppard, 1986) • DNA nanotechnology (Seeman, 2003). Implementation of abstract Wang- tiles with DNA tiles Winfree, 2003 The Tile Assembly Model • Only tiles with binding strength > 2 bonds will bind Advantages of Biomolecular Computation • Ultra Scale: each ”processor” is a molecule. • Massively Parallel: number of elements could be 1018 to 1020 • High Speed: perhaps 1015 operations per second. • Low Energy: – example calculation ~10-19 Joules/op. – electronic computers ~10-9 Joules/op. • Existing Biotechnology: well tested recombinant DNA techniques. Potential Disadvantages of Biomolecular Computation: • Many Laboratory Steps Required: – is very much reduced by Self-Assembly ! • Error Control is Difficult: – may use a number of methods for error- resilient Self-Assembly Error-Resilient Self-assembly • Bounds on error rates of self-assembly reactions: – No complete studies yet. – Non-computational assemblies appear to be less error-prone. • Methods that may Minimize Errors in self-assembly: – Annealing Temperature Variation. – Improved Sequence Specificity of DNA Annealing. – Step-wise Assembly versus Free Assembly. – Use of DNA Lattices as a Reactive Substrate for Error Repair. DNA and RNA Aptamers Selection of RNA and DNA aptamers that bind specifically to target proteins SELEX: SELEX Procedure for the Evolution of RNA Aptamers Binding the Receptors of Host-cell Matrix Molecules on Trypanosoma cruzi constant 40-nt random region constant T7-promoter 8 re-iterative rounds separation into cloning 5' 3' 3' 5' individual pools transcription 5' 3' 5' 3' 3' 5' RNA library containing 10 13 different sequences PCR amplification Aptamers 5' 3' RNA folding 5' reverse transcription laminin washing fibronectin displacement thrombospondin mixing heparan sulfate enriched RNA library target (trypomastigotes) Ulrich et al., Braz. J. Med. Biol. Res. 34, 295, 2001 Why to Use Nucleic Acids? Example for a biological active Nucleic acids form complex secondary RNA molecule (aptamer) and tertiary structures and bind with high affinity to their target proteins. They can be easily amplified using PCR techniques. DNA can be converted to RNA and RNA to DNA by in vitro transcription and reverse transcription procedures. Oligonucleotide polymers are excellent for in vivo studies as they can be chemically protected against enzymatically degradation. Oligonucleotides have a low immunogenic potential. 2´OH ribo- nucleotides Chemical modification of the 2OH position of the ribose of pyrimidines results in nuclease- resistance of the transcripts 2´amino ribo- nucleotides 2’ fluoro ribo- nucleotides What are the Possible Actions of Selected Aptamers on their Target Molecules (Enzymes or Receptors)? They can either acts: Inhibitors: by blocking the agonist binding site or by inducing a transition from an active to an inactive protein conformation Activators: by acting like an agonist or by stabilizing an active protein conformation Protectors: by binding to a regulatory site and not affecting protein function. Being biologically inactive, it will displace inhibitors from their binding sites and protect enzymes / receptors against inhibition Direct recognition Branden & Tooze, Introduction to Protein Structure, 1991 Sequence-specific because amino acid side chains H-bond with DNA base pairs in major groove. Structural basis well understood. Indirect recognition Example: Protein main-chain H- bonds with oligonucleotide backbone Branden & Tooze, Introduction to Protein Structure, 1991 sugar/phosphates Protein recognizes DNA / RNA structure Minor groove features Hydration spine DNA / RNAflexibility May be sequence specific Sequence determines structure The Use of SELEX •As an synthetic antibody to determine the concentration of target molecules in biological fluids •As an activator or inhibitor to study the functions of target proteins •To target intracellular proteins and establish stable knock-outs of these proteins •To determine the location of inhibitor / activator binding site on the target •To isolate and purify the target molecule •To evolve novel catalytic RNAs •To evolve stable aptamers for in vivo applications and therapy Aptamers Recognize their Target Proteins with the Same Specifity as Antibodies Western blot with anti- bradykinin B1 receptor antibody Western blot with aptamers selected against cell membranes containing B1 receptors 49 KDa 38 KDa 38 KDa 27 KDa w/o transf. control Re-iterative SELEX Rounds Result in Nanomolar Affinities of RNA Ligands to their Protein Targets -9 -8 Log dissociation constant (M) -7 -6 -5 -4 -3 -2 0 1 2 3 4 5 6 7 8 9 10 11 SELEX round Strategies for gene regulation by RNA sensors
"Self Organizing Bio structures NB2 2009 L Duroux Lecture 5 DNA Self Assembly Applications The trends in nano fabrication • The miniaturization to"