Self Organizing Bio structures NB2 2009 L Duroux Lecture 5 DNA Self Assembly Applications The trends in nano fabrication • The miniaturization to by wanghonghx

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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

								
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