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Playing with DNA as nanoscale construction

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Presentation on theme: "Playing with DNA as nanoscale construction"— Presentation transcript:

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.”

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