1. Playing with DNA as nanoscale construction
material – engineers go beyond biology
Other technologies for making things at this scale : e-beam lithograpy, scanning-tunneling microscopy, chemical self-assembly
Constructions we’ll talk about:
2-d DNA sheet with hexagonal array of ~30 nm holes
3-d DNA polyhedra ~10-40 nm diameter
“1-d” DNA tubes ~5-50nm diameter x several mm long
replicatable multi-strand DNA tubes

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

3. Holliday junction – natural intermediate in DNA
recombination: 2 inter-linked ds segments;
note single strands are flexible and can leave 1
double helix to join another

4. 2d-DNA tile arrays- He et al, JACS 127: 12202 (2005) Man-made version
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)
What are 5’->3’ sequences
of DNAs 1, 2 and 3?
Could you re-
engineer
this with
different
seq?

5. Overhangs are palindromes -> multimerization
Fig 1B in supplementary
material
Overhangs are palindromes
-> multimerization
CGCG
GCGC
CATG
GTAC

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

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

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

11. DNA structures analyzed by AFM, cryoEM and
dynamic light scattering (DLS)
DLS principle – if concentration of light scatterers is low,
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 ~l,
which is function of diffusion constant and particle r.

12. Measure how scattered light intensity changes over time
Time correlation function
<> = average over t
For monodisperse scatterers

13. DLS
AFM
Cryo-EM
<- Cryo-EM reconstructions
Tetrahedra

14. Dedecahedra

15. Buckeyballs

16. Summary
You can choose sequences so that short DNA pieces
self-assemble to form novel, porous, thin film
~100mm x 2nm (!) hexagonal lattices ~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, nm, 3D structures

17. More complicated hybridization structures when
single DNA strands bridge >1 helix
Tubes Yin et al, Science 321:824 (2008) 5’
3-helix ribbon
Arrows indicate 5’->3’ direction; #’s = length in bp;
letters (colors) denote particular sequence

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

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

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

21. How are adjacent helices positioned in a tube?
Looking down long axis of tubes:
What determines angles d10, d11 etc.?
d10

22. ~10.5 bases/turn of helix => ~3600 /10.5 = 34.30/base
complementary bases are from opp. strands and
are separated by ~2100 (not 1800, related to
major/minor groove asymmetry)
d10

23. d10
Angle between first base in U1 as it enters helix 2 and its complementary base in U2 is 2100; add 34.30/base x 11 bases in sequence a in strand U2 until next strand exits helix 2 => d11 = o x 11 – 360o = 470 (d10 = 130 if 10 bases in segment a).

24. They alternate segments of length 10 and 11 nt
-> d10 = 13o d11 = 47o <d> = 30o ->
~12 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?

25. 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
50nm

26. 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 which act as pushing/pulling motors and tracks for other protein motors to move along - could DNA tubes be engineered to have similar properties?

27. Paperby Seeman group in current Nature 478:225 (2011)
uses similar ideas to template self-replication of higher
order dna nanostructures
tile = set of intertwined double helices, e.g.
How many double helices in this tile?
What holds it together?

28. Can assemble tiles “longitudinally” via ss overhangs
Will these assemble? In what order?

29. Some of the tiles are designed with extra loop with biotin,
so that you can label with streptavidin and see it in AFM
Which
tiles bind
streptavidin?

30. This allows check if linear order of tiles is as expected

31. Now use short “up” and “down” overhangs to assemble
a row of complementary tiles in register

32. Use linker oligos to join newly assembled tiles in chain
Capture new tile assembly with streptavidin bead

33. Melt off template tile assembly
up and down links are only 7 bases long
so they melt at 370, while longitudinal links
are longer and stable at 370
Purify new linear assembly with magnet

34. Now assemble new string of tiles using daughter
string as template
They design so that granddaughter string is same
as initial string -> “self-replication”
Note how cludgy compared to natural DNA replication
but higher order nanostructure is replicated
using ideas and materials from biology

35. 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 double helices 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

36. DNA assemblies can be made dynamic
Basic idea – overhanging ss can be used as “toehold”
that allows added oligonucleotide to displace
a short piece of DNA in a double helix
Toehold displacement

37. Lots of papers in this area coming from Computer Sci.
Departments – language of “programmed” assembly,
abstract assembly notation (interdisciplinary cultural
issues!)
These papers are potential topics for term paper
presentations
This area seems to be searching for first “killer app.”