1. Can tools & materials of molecular biology be used to make nanoscale objects useful in non-biological applications? Other technologies for making things at this scale : e-beam lithograpy, scanning-tunneling microscopy, self-assembly Constructions we’ll talk about: 1.2-d DNA sheet  nm thick x  m’s on a side with a hexagonal array of  30 nm holes 2.3-d DNA polyhedra  10-40 nm diameter 3.“1-d” DNA tubes  5-50nm diameter x several  m long

2. DNA double helix 2nm 3.3nm 10 bp 1 2 4 5

3. Holliday junction – intermediate in DNA recombination 2 inter-linked ds segments; assoc with proteins in biological systems and transient, but don’t need protein and can be stable as man-made objects Questions not discussed: What are dimensions of gap? What is interplay between monomer dimensions, electrostatic repulsion, base pairing? How stiff is it? Is it planar?

4. Fig 1A, SOM ss region may allow arms to bend out of plane Arms are pairs of dbl helices fold backs may distort dbl helix 2d-DNA tile arrays- He et al, JACS 127: 12202 (2005)

5. Fig 1B, SOM Palindromic overhangs -> multimerization CGCG GCGC CATG GTAC

6. Fig 1 extra half turn ? -> alternating “up” vs “down” out of plane curvature * * ** * *

7. Gold metalized version of c shows inverse pattern Fig 4 ~2-4 nm thick “saran wrap”, with ~25 nm pores!

8. Questions you might be left with In more geometric detail, where do ss overhangs exit helices? Can star units buckle out of plane in both directions? How rigid are they? Did they test different inter-node distances to see how that affects planarity and ability to form large sheets? Does junction between ds regions stress or deform structures? Is it obvious or amazing that this strategy works?

9. Could single-strand region be used to guide attachment of other DNA-labeled objects in  30 nm array?

10. They say longer ss region in center (red) permits more bending out of plane 4 turns out of plane curvatures all in same direction Lower conc favors smaller # of tiles in polyhedron 3-d polyhedra He et al Nature 452:198 (2008)

11. DNA structures analyzed by AFM, cryoEM and dynamic light scattering (DLS) DLS principle – if # of light scatterers is not infinite, scattered light intensity will fluctuate in time since scatterers move, and sometimes scatterers will be positioned such that scattered rays constructively (destructively) interfere. Time scale of fluctuations will be related to time it takes particles to move  which is function of diffusion constant and particle r.

12. http://www.wyatt.com/theory/qels/ Measure how scattered light intensity changes over time Time correlation function <> = average over t For monodisperse scatterers

13. DLSAFMCryo-EM <- Cryo-EM reconstructions Tetrahedra

14. Dedecahedra

15. Buckeyballs

16. Summary DNA arrays can form novel, porous, thin films  100  m x 2nm (!) hexagonal lattice  25nm pores can be metallized ? mechanical, thermal, electric properties ? could be used as template to position objects  25nm scale; as novel filter DNA can make variety of closed, 10-60 nm, 3D structures

17. More complicated hybridization patterns when single DNA strands bridge >1 helix TubesYin et al, Science 321:824 (2008) 5’ 3-helix ribbon

18. Atomic Force Microscopy (AFM) used to analyze ribbons

19. 5-helix ribbon AFM images – width, pattern c/w model; straightness (rigidity, persistence length) increases with width 50nm 5-helix ribbon

20. 6-helix tube 12-helix tube Curvature model  need  12 helices to form unstressed tubes Take away edge strands, make “top” and “bottom” seq’s complementary, and ribbons roll into tubes!

21. Counter clockwise angle between any base (e.g. first base in U1 that hybridizes in helix 2) and its complementary base in a ds DNA is 210 0 ; add 34.3 0 /base in helix till U2 strand exits, subtract (modulo) 360 0 ->  k     x k – 360 o where k = # nt in segment “a”   Unstressed curvature model

22. They alternate segments of length 10 and 11 nt ->             helices would close without stress But they see only 6-helix tubes using 6 different U-segments. Why might they form? – kinetic trap; if tube starts to form, it would have to melt many base pairs to open, so trapped in local potential energy well Suggests tubes contain potential energy that might be tapped for some future use! Or is curvature model oversimplified, not taking into account distortion in helix at cross-over points?

23. 50nm How they measured # helices in tube circum.: measure width by AFM; assume tubes open and flat- ten due to electrostatic interaction with mica; width  3nm x # of helices

24. Potential uses – metallize and use as variable diameter conducting wires? model system for study of effect of structure on persistence length/other mechanical properties (class 5) structure similar to protein microtubules (class 9) which act as pushing/pulling motors and tracks for other protein motors to move along - could DNA tubes be engineered to have similar properties?

25. Summary – base pairing is simple principle that can be used to engineer pieces of DNA that bind to each other in precisely defined places Exchange of ss between different ds enables construction of complex, interwoven structures As engineers, you can go beyond what Nature provides Lots of inventive constructions we did not have time to discuss (DNA “origami”, 3-d sculptures, sorting flat tiles by shape using photolithographically patterned plates) = potential topics for student presentations

26. Next class – dynamic DNA assemblies Basic idea – overhanging ss can be used as “toehold” to displace a short piece of DNA in a double helix Toehold displacement

27. Lots of papers in this area coming from Computer Sci. Departments – language of “programmed” assembly, abstract assembly notation (interdisciplinary cultural issues!) Read Yin, Nature paper on exponential displacement scheme Think about how it compares to pcr Bath and Turberfield review of DNA Nanomachines If interested, Seeman paper on DNA walker