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:

1. 2-d DNA sheet with 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 mm

long4. replicatable multi-strand DNA tubes

DNA double helix

2nm

3.3nm 10 bp

124

5

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 mayallow arms to bendout of plane

Arms are pairsof dbl helices

fold backs maydistort dbl helix

2d-DNA tile arrays-He et al, JACS 127:12202 (2005)

Man-made version

What are 5’->3’ sequences of DNAs 1, 2 and 3?Could you re-engineerthis withdifferent seq?

Fig 1B in supplementarymaterial

Overhangs are palindromes -> multimerization

CGCGGCGCCATGGTAC

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

*

*

* *

*

*

Gold metalized version of c showsinverse 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? 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 deform structures?

Is it obvious or amazing that this strategy works?

Could single-strand region be used to guide attachmentof other DNA-labeled objects in ~30 nm array?

They say longer ss region in center (red)permits more bending out of plane

4 turnsout of plane curvatures all in same direction

Lower conc favorssmaller # oftiles in polyhedron

3-d polyhedraHe et al Nature452:198 (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 sincescatterers 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 ~ , lwhich is function of diffusion constant and particle r.

http://www.wyatt.com/theory/qels/

Measure how scattered light intensity changes over timeTime correlation function

<> = average over t

For monodisperse scatterers

DLS AFM Cryo-EM

<- Cryo-EM reconstructions

Tetrahedra

Dedecahedra

Buckeyballs

Summary

You can choose sequences so that short DNA pieces self-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 filter

DNA can make variety of closed, 10-60 nm, 3D structures

More complicated hybridization structures whensingle 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

Atomic Force Microscopy (AFM) used to analyze ribbons

5-helix ribbon

AFM images – width, pattern c/w model; straightness (rigidity, persistence length) increases with width

50nm

5-helix ribbon

6-helix tube

12-helix tube

Curvature modelsuggests ~12 heliceswould form unstressed tubes

Take away edge strands, make “top” and “bottom” seq’scomplementary, -> ribbons roll into tubes!

d10

How are adjacent helices positioned in a tube?

Looking down long axis of tubes:

What determines angles d10, d11 etc.?

d10

~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)

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 = 2100 + 34.3o x 11 – 360o - 1800 = 470 (d10 = 130 if 10 bases in segment a).

d10

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

50nm

How they measured # helices intube circum.:measure widthby AFM; assume tubesopen and flat-ten due toelectrostaticinteraction 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 higherorder 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 bindstreptavidin?

This allows check if linear order of tiles is as expected

Now use short “up” and “down” overhangs to assemblea 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 370, while longitudinal linksare longer and stable at 370

Purify new linear assembly with magnet

Now assemble new string of tiles using daughterstring as template

They 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

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 culturalissues!)

These papers are potential topics for term paperpresentations

This area seems to be searching for first “killer app.”