In the name of GOD

1. In the name of GOD

2. Introduction to self-assembling DNA nanostructures Zeinab Mokhtari 1-Dec-2010

3. DNA is an excellent nanoconstruction material because of its inherent merits: • The rigorous Watson-Crick base pairing makes the hybridization between DNA strands highly predictable. • The structure of the B-form DNA double helix is well-understood. • DNA possesses combined structural stiffness and flexibility. The rigid DNA double helices can be linked by relatively flexible single-stranded DNA (ssDNA) to build stable motifs with desired geometry. • Modern organic chemistry and molecular biology have created a rich toolbox for readily synthesizing, modifying, and replicating DNA molecules. • DNA is a biocompatible material, making it suitable for the construction of multicomponent nanostructures made from heterobiomaterials.

4. Molecular-scale devices using DNA nanostructures have been engineered to have various capabilities : • execution of molecular-scale computation • use as scaffolds or templates for the further assembly of other materials (such as scaffolds for various hybrid molecular electronic architectures or perhaps high-efficiency solar-cells) • robotic movement and molecular transport • exquisitely sensitive molecular detection and amplification of single molecular events • transduction of molecular sensing to provide drug delivery

5. DNA Nanotechnology DNA Nanotechnology and its use to Assemble Molecular-Scale Devices unique advantages: Self-assembly Easy to design Fairly predictable Experimentally implemented A DNA (RNA) nanostructure is a multimolecular (supramolecular) complex consisting of a number of ssDNA that have partiallyhybridized, as designed, along their sub-segments.

7. DR. NADRIAN C. SEEMAN Chicago in 1945 B.S. in biochemistry from the University of Chicago in 1966 Ph.D from the Department of Crystallography at the University of Pittsburgh in 1970. He is most noted for his development of the concept of DNA nanotechnology beginning in the early 1980‘s. In fall 1980, while at a campus pub, Seeman was inspired by the M. C. Escher woodcut Depth to realize that a three-dimensional lattice could be constructed from DNA. He realized that this could be used to orient target molecules, simplifying their crystallographic study by eliminating the difficult process of obtaining pure crystals. In pursuit of this goal, Seeman's laboratory published the synthesis of the first three-dimensional nanoscale object, a cube made of DNA, in 1991. This work won the 1995 Feynman Prize in Nanotechnology. The concepts of DNA nanotechnology later found further applications in DNA computing, DNA nanorobotics, and self-assembly of nanoelectronics. He shared the Kavli Prize in Nanoscience 2010 with Donald Eigler “for their development of unprecedented methods to control matter on the nanoscale”. The goal of demonstrating designed three-dimensional DNA crystals was achieved by Seeman in 2009, nearly thirty years after his original elucidation of the idea.

8. more complicated nanostructures(branched DNA motifs) The simplest example of DNA self-assembly can be found in almost all living forms in nature: two complementary ssDNA molecules spontaneously hybridize together and form a double-stranded DNA (dsDNA) molecule. molecular biology : genecloning (the genomic DNA of interest)

9. useful types of motifs in DNA nanostructures :Stem-Loops and Sticky Ends visualizing programmed patterning on DNA nanostructures combining two DNA nanostructures together via hybridization of their complementary ssDNA

10. Holliday junctions to tie together various parts of a DNA nanostructure.

11. DNA tiles and lattices Self-assembly is a hierarchical process.

12. DNA scaffolds

13. DNA nanotubes

14. DNA origami

15. DNA cage

16. DNA box Aarhus University Center for DNA Nanotechnology

17. Thermal Annealing For experimental synthesis of the DNA nanostructure, the oligonucleotides with designated sequences are synthesized by a DNA synthesizer, purified via electrophoresis or chromatography, mixed together at the stoichiometric molar ratio in a near-neutral buffer containing divalent cations (usually Mg2+), heated to denature, and then gradually cooled to allow the ssDNA molecules to find their correct partners and adopt the most energy favorable conformation (i.e., self-assembly).

18. Fluorescent DNA Nanotags Fluorescent DNA Nanotags Based on a Self-Assembled DNA Tetrahedron nn800727x ACSNANO VOL.3 NO.2 425–433 2009

19. Fluorescent labeling of biomolecules • fast signal acquisition • high sensitivity • suitability for multiplex assaying by using fluorophores that emit at different wavelengths

20. multichromophore arrays Arrangement of multiple dye molecules into arrays • improves the efficiency of light absorption • provides efficient energy transfer pathways, allowing fine-tuning of the emission wavelength positioning the fluorophores at predefined spatial positions that prevent self-quenching but promote energy transfer

21. loading with fluorescent intercalating dyes The inherent steric constraints imposed by the DNA double helix restrict the placement of intercalating dyes to distances and orientations that prevent self-quenching, yet the compact structure of the nanotag allows efficient energy transfer to remote acceptor groups, resulting in bright multichromophore assemblies termed as “DNA nanotags”. • higher density of fluorophores without significantly compromised brightness • improved photostability • good resistance to degradation by nuclease enzymes

22. four oligonucleotide strands with partially complementary sequences can self-assemble into a tetrahedron consisting of 102 Watson-Crick base pairs. compact 3-dimensional assembly based on a tetrahedron (TH) nanostructure Nanotag Design 8.5 nm edges : 17 base pairs in length The binding sites for the light-harvesting pintercalating dyes 8 intercalating chromophores bind to the 17 bp-long edge “hinges” consisting of two unpaired nucleotides that ensure sufficient flexibility to form the tetrahedron bisintercalating dimer of oxazole yellow: high affinity for double-stranded DNA, high extinction coefficient of 98900M-1 cm-1, and fluorogenic properties

23. Nanotags Linear (1D) Branched 3-way junction (2D) Tetrahedron (3D) (the compact nature of the TH)

24. Nanotag Assembly and Characterization neighbor exclusion principle : one intercalator for every two base pairs due to steric constraints imposed by the DNA Saturation : half of the binding sites are occupied odd number of base pairs in each edge and the symmetry of the tetrahedron nanostructure → only 4 bisintercalators bind per edge, giving 48 total intercalated chromophores. DNA nanostructures : thermal annealing process

25. Cooperative melting transitions in the presence of the dye The melting temperature of a DNA helix is the temperature at which half of all the molecules are fully hybridized as double helix, while the other half are single stranded. a broad low temperature transition in the absence of YOYO-1 Intercalation of YOYO-1 increases the thermal stability of the DNA structures by approximately the same amount, regardless of the dimensionality of the nanostructure. The DNA-dye nanostructures are fully assembled at room temperature.

26. significant differences in fluorescence intensities of the intercalator dye in the presence of the three nanostructures similar sequence composition (47-48% GC) for DNA templates The difference in fluorescence intensity is unlikely to be due to the known sequence dependence of fluorescence quantum yield for this dye. • one or more of the chromophores • insert into the junction regions linking different helices in the 2- and 3-D structures → not as constrained as a fully intercalated Dye → lower quantum yield • traps for energy migrating through the assembly Intermediate weaker

27. Energy Transfer Experiments In flow cytometry and fluorescence microscopy, it is useful to have labels that fluoresce in different colors but that can be excited at the same wavelength, such that different populations can be distinguished while using the same light source. helix junction points multicolor labeling highly efficient ET from intercalated YOYO-1 to terminal acceptor dyes (Cy3 or WellRed-D2) attaching 1-4 Cy3 acceptor dyes to the 5’-termini of the DNA strands

28. The ET efficiency increases monotonically with the number of Cy3 acceptors, reaching a maximum of 95%. The compact nature of the TH, which localizes all intercalated dye molecules within a diameter of less than 10 nm, similar to the critical transfer distance of 7.3 nm for the YOYO-1/Cy3 pair.

29. energy migration among the cointercalatedchromophores particularly within a given edge The TH acts as an antenna to harvest light with very strong efficiency and transfers the excitation to a lower energy acceptor dye at a nearby helix junction. TH nanotags with and without the Cy3 acceptor dyes can be used to label two different species of interest, while being excitable at the same wavelength.

30. ACSNANO VOL.3 NO.2 425–433 2009 Biochemistry Volume 48, Number 8 March 3, 2009 Bio-inspired and Nano-scale Integrated Computing Wiley, USA, (2007).

31. Thanks