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

• An example of a reciprocal exchange: Two DNA helices are connected by sharing two DNA strands (Seeman, 2001)

Oligonucleotides

A BC

D

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

• 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

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

()– Distance at which

potential is zero ()

• 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

• DNA density of 14000 molecules / sphere lead to unstructured aggregates

• DNA density of 3700 molecules / sphere lead to self-assembled crystallites

14000/sphere 3700/sphere

3700/sphere T >> Tm

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

Homologous duplexesReciprocal exchange

Dyad Axis of seq. symmetry

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

A

B

C

Example with triangle motifs

Central core strands

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

Yan et al, 2003. Science 301:1882-84

4x4 DNA tile

DNA-Templated Self-Assembly ofMetallic Nanocomponent Arrays on a

Surface

DNA-Templated Self-Assembly ofMetallic Nanocomponent Arrays on a

Surface

DNA-Templated Self-Assembly ofMetallic Nanocomponent Arrays on a

Surface

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 tilesStreptavidin

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 BiomolecularComputation:

• 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

con stant T 7 -p r o m o te r

1 3

5 '

5 '

5 '5 '

3 '

3 '

3 '

3 '

3 '

3 '

5 '

5 '5 '

40-nt rand om reg ion

R N A lib rary con tain in g 10 d ifferen t seq uences

R N A fo ld in g

tran scr ip tion

m ix in g

w a sh in gd isp la cem en t

lam in in

re v e rse tr a n scr ip tio n

P C R am p lifica tion

8 re -itera tive ro u n d s sep aration in to in d iv id u a l p o o ls

c lo n in g

A p ta m ers

fib ron ectin

throm bo spond in

h ep aran su lfa te

enr ich ed R N A lib ra rytarget(trypom astigo tes)

con stant

Ulrich et al., Braz. J. Med. Biol. Res. 34, 295, 2001

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 vitrotranscription 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)

F

2´OH ribo-nucleotides

2´amino ribo-nucleotides

2’ fluoro ribo-nucleotides 0

20

40

60

80

100

120

0 10 20 30 40 50

Unmodified RNA2´aminopyrimidine modified RNA

Incubation with serum in hours

Chemical modification of the 2OH position of the ribose of pyrimidines results in nuclease-resistance of the transcripts

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

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

Indirect recognition

Branden & Tooze, Introduction to Protein Structure, 1991

Protein recognizes DNA / RNA structureMinor groove featuresHydration spineDNA / RNAflexibility

May be sequence specificSequence determines structure

Example:

Protein main-chain H-bonds with oligonucleotide backbone sugar/phosphates

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

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

-9

-8

-7

-6

-5

-4

-3

-2

0 1 2 3 4 5 6 7 8 9 10 11

Lo

g d

iss

oc

iati

on

co

ns

tan

t (M

)

SELEX round

Re-iterative SELEX Rounds Result in NanomolarAffinities of RNA Ligands to their Protein Targets

Strategies for gene regulation by RNA sensors