Presentation on theme: "Playing with DNA as nanoscale construction"— Presentation transcript:
1 Playing with DNA as nanoscale construction material – engineers go beyond biologyOther technologies for making things at this scale : e-beam lithograpy, scanning-tunneling microscopy, chemical self-assemblyConstructions we’ll talk about:2-d DNA sheet with hexagonal array of ~30 nm holes3-d DNA polyhedra ~10-40 nm diameter“1-d” DNA tubes ~5-50nm diameter x several mm longreplicatable multi-strand DNA tubes
2 DNA double helix3.3nm10 bpminorgroovemajorgroove51422nm
3 Holliday junction – natural intermediate in DNA recombination: 2 inter-linked ds segments;note single strands are flexible and can leave 1double helix to join another
4 2d-DNA tile arrays- He et al, JACS 127: 12202 (2005) Man-made version ss region mayallow arms to bendout of planeArms are pairsof dbl helicesfold backs maydistort dbl helix2d-DNA tile arrays-He et al, JACS 127:12202 (2005)What are 5’->3’ sequencesof DNAs 1, 2 and 3?Could you re-engineerthis withdifferentseq?
5 Overhangs are palindromes -> multimerization Fig 1B in supplementarymaterialOverhangs are palindromes-> multimerizationCGCGGCGCCATGGTAC
6 ******extra half turn ? -> alternating “up”vs “down” out of plane curvatureFig 1
7 ~2-4 nm thick “saran wrap”, with ~25 nm pores! Gold metalizedversion of c showsinverse patternFig 4
8 Questions you might be left with In more geometric detail, where do ss overhangs exithelices?Can star units buckle out of plane in both directions? Howrigid are they?Did they test different inter-node distances to see howthat affects planarity and ability to form large sheets?Does junction between ds regions stress or deformstructures?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 planeLower concfavorssmaller # oftiles inpolyhedron3-d polyhedraHe et al Nature452:198 (2008)4 turnsout of plane curvaturesall 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 sincescatterers move, and sometimes scatterers will bepositioned such that scattered rays constructively(destructively) interfere. Time scale of fluctuationswill 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 tFor monodisperse scatterers
16 SummaryYou can choose sequences so that short DNA piecesself-assemble to form novel, porous, thin film~100mm x 2nm (!) hexagonal lattices ~25nm porescan be metallized? mechanical, thermal, electric properties? could be used as template to position objects~25nm scale; as novel filterDNA can make variety of closed, nm, 3D structures
17 More complicated hybridization structures when single DNA strands bridge >1 helixTubes Yin et al, Science 321:824 (2008)5’3-helix ribbonArrows indicate 5’->3’ direction; #’s = length in bp;letters (colors) denote particular sequence
18 Atomic Force Microscopy (AFM) used to analyze ribbons
20 Take away edge strands, make “top” and “bottom” seq’s complementary, -> ribbons roll into tubes!6-helix tube12-helix tubeCurvature modelsuggests ~12 heliceswould formunstressed 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 andare separated by ~2100 (not 1800, related tomajor/minor groove asymmetry)d10
23 d10Angle 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 stressBut they see only 6-helix tubes using 6 different U-segments.Why might they form? – kinetic trap; if tube startsto form, it would have to melt many base pairsto open, so trapped in local potential energy wellSuggests tubes contain potential energy that mightbe tapped for some future use!Or is curvature model oversimplified, not taking intoaccount distortion in helix at cross-over points?
25 How they measured # helices in tube circum.: measure width by AFM; assume tubesopen and flat-ten due toelectrostaticinteractionwith mica;width ~3nm x# of helices50nm
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 higherorder dna nanostructurestile = 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 AFMWhichtiles bindstreptavidin?
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 longso they melt at 370, while longitudinal linksare longer and stable at 370Purify new linear assembly with magnet
34 Now assemble new string of tiles using daughter string as templateThey design so that granddaughter string is sameas initial string -> “self-replication”Note how cludgy compared to natural DNA replicationbut higher order nanostructure is replicatedusing ideas and materials from biology
35 Summary –base pairing is simple principle that can be usedto engineer pieces of DNA that bind to eachother in precisely defined placesExchange of ss between different double helices enablesconstruction of complex, interwoven structuresAs engineers, you can go beyond what Nature providesLots of inventive constructions we did not have timeto discuss (DNA “origami”, 3-d sculptures, sortingflat tiles by shape using photolithographically patternedplates) = 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 displacea short piece of DNA in a double helixToehold displacement
37 Lots of papers in this area coming from Computer Sci. Departments – language of “programmed” assembly,abstract assembly notation (interdisciplinary culturalissues!)These papers are potential topics for term paperpresentationsThis area seems to be searching for first “killer app.”
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