Presentation on theme: "Playing with DNA as nanoscale construction material – engineers go beyond biology Other technologies for making things at this scale : e-beam lithograpy,"— Presentation transcript:
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 well talk about: 1.2-d DNA sheet with hexagonal array of 30 nm holes 2.3-d DNA polyhedra nm diameter 3.1-d DNA tubes 5-50nm diameter x several m long 4.replicatable multi-strand DNA tubes
DNA double helix 2nm 3.3nm 10 bp major groove minor groove
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
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: (2005) Man-made version What are 5->3 sequences of DNAs 1, 2 and 3? Could you re- engineer this with different seq?
Fig 1B in supplementary material Overhangs are palindromes -> multimerization CGCG GCGC CATG GTAC
Fig 1 extra half turn ? -> alternating up vs down out of plane curvature * * ** * *
Gold metalized version of c shows inverse pattern Fig 4 ~2-4 nm thick saran wrap, with ~25 nm pores!
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?
Could single-strand region be used to guide attachment of other DNA-labeled objects in 30 nm array?
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)
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 which is function of diffusion constant and particle r.
Measure how scattered light intensity changes over time Time correlation function <> = average over t For monodisperse scatterers
Summary You can choose sequences so that short DNA pieces self-assemble to form novel, porous, thin film 100 m 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
More complicated hybridization structures when single DNA strands bridge >1 helix TubesYin et al, Science 321:824 (2008) 5 3-helix ribbon Arrows indicate 5->3 direction; #s = length in bp; letters (colors) denote particular sequence
Atomic Force Microscopy (AFM) used to analyze ribbons
6-helix tube 12-helix tube Curvature model suggests 12 helices would form unstressed tubes Take away edge strands, make top and bottom seqs complementary, -> ribbons roll into tubes!
How are adjacent helices positioned in a tube? Looking down long axis of tubes: What determines angles 10, 11 etc.?
10.5 bases/turn of helix => /10.5 = /base complementary bases are from opp. strands and are separated by (not 180 0, related to major/minor groove asymmetry)
Angle between first base in U1 as it enters helix 2 and its complementary base in U2 is ; add /base x 11 bases in sequence a in strand U2 until next strand exits helix 2 => 11 x 11 – 360 o = 47 0 ( if 10 bases in segment a).
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?
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
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?
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?
Will these assemble? In what order? Can assemble tiles longitudinally via ss overhangs
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?
This allows check if linear order of tiles is as expected
Now use short up and down overhangs to assemble a row of complementary tiles in register
Use linker oligos to join newly assembled tiles in chain Capture new tile assembly with streptavidin bead
Melt off template tile assembly up and down links are only 7 bases long so they melt at 37 0, while longitudinal links are longer and stable at 37 0 Purify new linear assembly with magnet
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
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
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
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.