1. Self-Organizing Bio-structures NB2-2009 L.Duroux

2. Lecture 5: DNA Self-Assembly Applications

3. 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

4. Remember Nucleic Acids (DNA) and their Self-Assembly properties An example of a reciprocal exchange: Two DNA helices are connected by sharing two DNA strands (Seeman, 2001) Oligonucleotides AB C D

5. 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

6. DNA as scaffold for nano- architectures

7. 1. Using ssDNA as template to self-assemble nanostructures

8. A simple case of ssDNA-functionalized micro-beads 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 T= 23¤C T= 50¤C Polymer brush -> steric repulsion Valignat et al, 2005. PNAS 102(12): 4225-29

9. Interaction Energies of micro-beads Trick is: create a U minimum well outside U vdW well Balancing finely U rep and U dna Limiting the number of base-pair bonds between two cDNAs

10. Lennard-Jones Potential Potential function of: –Depth of potential well (  ) –Distance at which potential is zero (  ) Term in power 12 describes repulsive forces

11. 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

12. 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

13. Binding Energies as function of rigidity of ssDNA For identical Tm (43.7¤C), “rigid” spacer gives stronger U well

14. Effect of ssDNA density on aggregate structuration DNA density of 14000 molecules / sphere lead to unstructured aggregates DNA density of 3700 molecules / sphere lead to self-assembled crystallites 14000/sphere3700/sphere T >> Tm

15. 2. DNA tiles: the ”building bricks”

16. 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

17. Branch molecules and branch migration Homologous duplexes Reciprocal exchange Dyad Axis of seq. symmetry

18. Stable branch junction No Axis of seq. symmetry No complement sequence in corners

19. Stem formation on inexact complementary strands

20. Creation of stable motifs with DNA by reciprocal exchange

21. Combinatorial self-assembly of DNA nanostructures

22. AFM pictures of DNA tiles combinations

23. Topology measurements by AFM

24. Motif formed by quadruple cross-over (QX) & Lattice A B

25. The concept of DNA tiles A B C Example with triangle motifs Central core strands Side strands Horseshoe strands

26. Lattices from SA of triangle motifs Brun et al, 2006

27. Creation of 3D tiles with QX motifs A B C

28. 3D structures from DNA self-assembly (Seeman, 2003) A cube A truncated octahedron

29. Another tiling process using tecto-squares Chworos et al., Science306, 2068 (2004).

30. 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.

31. DNA as template for electrical nano-wires A step toward “nano-electronics”

32. DNA for Molecular Lithography: principle Gazit, 2007. FEBS J. 274:317-322

33. DNA lithography: towards nanoelectronics Niemeyer, 2002. Science, 297:62-63.

34. Conducting DNA-nanowires Yan et al, 2003. Science 301:1882-84 4x4 DNA tile

35. DNA-Templated Self-Assembly of Metallic Nanocomponent Arrays on a Surface

38. Templated array of proteins on 4x4 nanogrids In nano-electronics designs: possibility to self-assemble proteins on DNA grid  Nano-electronics components Biotinylated DNA 4x4 tiles Streptavidin

39. Metallization and conductivity measurements of DNA 4x4 tile ribbons 500 nm

40. Programmable Self-Assembly of DNA

41. 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).

42. Molecular-scale pattern for RAM-memory

43. 3 components for DNA computing DNA computing (Adleman, 1994) Theory of tilings (Grunbaum and Sheppard, 1986) DNA nanotechnology (Seeman, 2003).

44. Implementation of abstract Wang- tiles with DNA tiles Winfree, 2003

45. The Tile Assembly Model Only tiles with binding strength > 2 bonds will bind

46. Advantages of Biomolecular Computation Ultra Scale: each ”processor” is a molecule. Massively Parallel: number of elements could be 10 18 to 10 20 High Speed: perhaps 10 15 operations per second. Low Energy: –example calculation ~10-19 Joules/op. –electronic computers ~10-9 Joules/op. Existing Biotechnology: well tested recombinant DNA techniques.

47. 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

48. 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.

49. DNA and RNA Aptamers Selection of RNA and DNA aptamers that bind specifically to target proteins

50. SELEX:

51. SELEX Procedure for the Evolution of RNA Aptamers Binding the Receptors of Host-cell Matrix Molecules on Trypanosoma cruzi Ulrich et al., Braz. J. Med. Biol. Res. 34, 295, 2001

52. Why to Use Nucleic Acids? Nucleic acids form complex secondary 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. Example for a biological active RNA molecule (aptamer)

53. 2´OH ribo- nucleotides 2´amino ribo- nucleotides 2’ fluoro ribo- nucleotides Chemical modification of the 2OH position of the ribose of pyrimidines results in nuclease- resistance of the transcripts

54. 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

55. Sequence-specific because amino acid side chains H-bond with DNA base pairs in major groove. Structural basis well understood. Direct recognition Branden & Tooze, Introduction to Protein Structure, 1991

56. Indirect recognition Branden & Tooze, Introduction to Protein Structure, 1991 Protein recognizes DNA / RNA structure Minor groove features Hydration spine DNA / RNAflexibility May be sequence specific Sequence determines structure Example: Protein main-chain H- bonds with oligonucleotide backbone sugar/phosphates

57. 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

58. Aptamers Recognize their Target Proteins with the Same Specifity as Antibodies 49 KDa 38 KDa 27 KDa 38 KDa Western blot with aptamers selected against cell membranes containing B1 receptors w/o transf. control Western blot with anti- bradykinin B1 receptor antibody

59. -9 -8 -7 -6 -5 -4 -3 -2 01234567891011 L o g d i s s o c i a t i o n c o n s t a n t ( M ) SELEX round Re-iterative SELEX Rounds Result in Nanomolar Affinities of RNA Ligands to their Protein Targets

60. Strategies for gene regulation by RNA sensors