The ionic conductivity and structural stability

of DNA origami

Chen-Yu Li 2014/7/7

2

Content

Introduction

Results:

(1) DNA origami expands under electric field

(2) [Mg2+] dependence of conductivity

(3) Voltage dependence of conductivity

Summary

3

fraction of scaffold strands that were incorporated into monomericspecies after folding varied from 7% to 44% for these targets asestimated by ethidium-bromide fluorescence intensity. Gel-purifiedparticles were generally observed to be monodisperse with a homo-genous shape (Fig. 2f); defect analysis for a series of related objectscan be found elsewhere21.

The five objects displayed in Fig. 2 demonstrate the generality of thishoneycomb-pleated origami approach in approximating variousthree-dimensional shapes. Figure 2a shows a structure resembling amonolith, assembled in the form of a honeycomb-pleated block as inFig. 1, except with ten layers instead of three. Particles display thepredicted pattern of holes and stripes consistent with a honeycomblattice of cylinders. Figure 2b shows a square nut, the cross-section ofwhich is a block of the honeycomb lattice with an internal pore shapedlike a six-pointed star. Figure 2c shows a structure that resembles abridge with hand rails. This shape demonstrates that different cross-section patterns can be implemented along the helical axis. Figure 2dshows a slotted cross, a structure composed of two honeycomb-lattice-based domains that sit at 90u to one another. One domain isH-shaped, the other is O-shaped. The centre of the H-domain passesthrough the slot of the O-domain, and the two domains are connectedby a pair of Holliday-junction crossovers derived from the scaffoldstrand. The 90u angle between domains is enforced by steric collisionsbetween the ends of helices on the H-domain and the sides of heliceson the O-domain. The fifth particle image for the slotted cross inFig. 2d shows a defective particle, where the slot in the O-domaincan be seen clearly. Figure 2e shows a stacked cross, where againtwo domains sit at 90u to one another. One domain is C-shaped,the other domain resembles a pod with a cavity. The pod domainconsists of four sub-modules that are each connected to theC-shaped domain by a Holliday-junction crossover derived fromthe scaffold strand. Upon folding, the sub-modules connect to eachother by staple linkages, enforcing a rotation to yield the complete poddomain oriented 90u to the C-module.

For the monolith, an effective diameter of 2.4 nm (60.1 nm standarddeviation, s.d.) per individual double helix was observed (Fig. 2g, h),while for the square nut an effective diameter of 2.1 nm (60.1 nm s.d.)per individual double helix was observed (Fig. 2i, j). Assuming anunhydrated helical diameter of 2.0 nm (although the hydrodynamic

helical diameter has been estimated22 as 2.2–2.6 nm), this observationsuggests the presence of inter-helical gaps produced by electrostaticrepulsion8 of the order of 0.1–0.4 nm, significantly less than the1.0 nm gap size estimated for Rothemund flat origami. This discre-pancy is probably related to the roughly twofold higher density ofcrossovers present in the honeycomb-pleated origami. Differences ineffective helix diameter between architectures may originate in partfrom staining artefacts (for example, cavities where large amounts ofpositively charged stain accumulate, or flattening).

Three key determinants for folding of honeycomb-pleated origamiwere investigated: duration of thermal ramp, divalent-cation concen-tration, and monovalent-cation concentration. Folding with shortthermal ramps (Fig. 3b, lefthand lanes), low concentrations of MgCl2(Fig. 3d, lefthand lanes), or high concentrations of NaCl (Fig. 3f,lefthand lanes) yielded a slowly migrating species upon agarose-gelelectrophoresis and grossly misshapen objects as observed by transmis-sion electron microscopy (for example, see Fig. 3c). In contrast, week-long thermal annealing at higher concentrations of MgCl2 combinedwith low concentrations of NaCl yielded a fast-migrating species uponagarose-gel electrophoresis and well-folded particles as observed byelectron microscopy (Fig. 3e), along with lower mobility bands corres-ponding to multimerized and aggregated objects. The apparent trendwas that increasing agarose-gel mobility correlated with improvementof quality of folding as observed by transmission electron microscopy,suggesting that correctly folded structures tend to be more compactthan misfolded versions.

Divalent cations thus appear to accelerate the rate of proper foldingand increase the amount of undesired aggregation whereas monovalentcations appear to decelerate the rate of proper folding and decrease theamount of undesired aggregation. Many of the structures require week-long thermal ramps for proper folding, even under idealized divalent-and monovalent-cation concentrations. Divalent cations may accel-erate target folding by specific stabilization of Holliday-junction cross-overs23 and by nonspecific stabilization of compact DNA24 foldingintermediates, although they may also stabilize nontarget aggregatesby a similar mechanism. Monovalent-cation binding might competewith divalent-cation binding, and thereby antagonize both targetcompaction and nontarget aggregation, analogous to how such bind-ing inhibits multivalent-cation-induced DNA condensation25. Folding

= =

zx

y

i

i

ii

iii

ii

iii

iii

i ii iii i ii

a b

dc

Figure 1 | Design of three-dimensional DNA origami. a, Double helicescomprised of scaffold (grey) and staple strands (orange, white, blue) runparallel to the z-axis to form an unrolled two-dimensional schematic of thetarget shape. Phosphate linkages form crossovers between adjacent helices,with staple crossovers bridging different layers shown as semicircular arcs.b, Cylinder model of a half-rolled conceptual intermediate. Cylinders

represent double helices, with loops of unpaired scaffold strand linking theends of adjacent helices. c, Cylinder model of folded target shape. Thehoneycomb arrangement of parallel helices is shown in cross-sectional slices(i–iii) parallel to the x–y plane, spaced apart at seven base-pair intervals thatrepeat every 21 base pairs. All potential staple crossovers are shown for eachcross-section. d, Atomistic DNA model of shape from c.

NATURE | Vol 459 | 21 May 2009 LETTERS

415 Macmillan Publishers Limited. All rights reserved©2009

© 2006 Nature Publishing Group

y-direction. As noticed before in DNA lattices15, parallel helices insuch structures are not close-packed, perhaps owing to electrostaticrepulsion. Thus the exact y-resolution depends on the gap betweenhelices. The gap, in turn, appears to depend on the spacing ofcrossovers. In Fig. 1a crossovers occur every 1.5 turns along alter-nating sides of a helix, but any odd number of half-turnsmay be used.In this study, data are consistent with an inter-helix gap of 1 nmfor 1.5-turn spacing and 1.5 nm for 2.5-turn spacing, yielding ay-resolution of 6 or 7 nm, respectively.Conceptually, the second step (illustrated in Fig. 1b) proceeds by

folding a single long scaffold strand (900 nucleotides (nt) in Fig. 1b)back and forth in a raster fill pattern so that it comprises one of thetwo strands in every helix; progression of the scaffold from one helixto another creates an additional set of crossovers, the ‘scaffoldcrossovers’ (indicated by small red crosses in Fig. 1b). The funda-mental constraint on a folding path is that the scaffold can form acrossover only at those locations where the DNA twist places it at a

tangent point between helices. Thus for the scaffold to rasterprogressively from one helix to another and onto a third, the distancebetween successive scaffold crossoversmust be an odd number of halfturns. Conversely, where the raster reverses direction vertically andreturns to a previously visited helix, the distance between scaffoldcrossovers must be an even number of half-turns. Note that thefolding path shown in Fig. 1b is compatible with a circular scaffoldand leaves a ‘seam’ (a contour which the path does not cross).Once the geometric model and a folding path are designed, they

are represented as lists of DNA lengths and offsets in units of half-turns. These lists, along with the DNA sequence of the actual scaffoldto be used, are input to a computer program. Rather than assuming10.5 base pairs (bp) per turn (which corresponds to standard B-DNAtwist), the program uses an integer number of bases between periodiccrossovers (for example, 16 bp for 1.5 turns). It then performs thethird step, the design of a set of ‘staple strands’ (the coloured DNAstrands in Fig. 1c) that provide Watson–Crick complements for the

Figure 1 |Design of DNAorigami. a, A shape (red) approximated by paralleldouble helices joined by periodic crossovers (blue). b, A scaffold (black) runsthrough every helix and forms more crossovers (red). c, As first designed,most staples bind two helices and are 16-mers. d, Similar to c with strandsdrawn as helices. Red triangles point to scaffold crossovers, black triangles toperiodic crossovers with minor grooves on the top face of the shape, bluetriangles to periodic crossovers with minor grooves on bottom. Cross-sections of crossovers (1, 2, viewed from left) indicate backbone positions

with coloured lines, andmajor/minor grooves by large/small angles betweenthem. Arrows in c point to nicks sealed to create green strands in d. Yellowdiamonds in c and d indicate a position at which staples may be cut andresealed to bridge the seam. e, A finished design after merges andrearrangements along the seam. Most staples are 32-mers spanning threehelices. Insets show a dumbbell hairpin (d) and a 4-T loop (e), modificationsused in Fig. 3.

ARTICLES NATURE|Vol 440|16 March 2006

298

DNA origami

Rothemund, P.W., 2006, Folding DNA to create nanoscale shapes and patterns, Nature, 440(7082), pp. 297-302. Douglas, S.M., Dietz, H., Liedl, T., Högberg, B., Graf, F. & Shih, W.M., 2009, Self-assembly of DNA into nanoscale three-dimensional shapes, Nature, 459(7245), pp. 414-8.

Scaffold: long ssDNA Staple: short (17~50 bp) ssDNA, connecting different parts.

The hybrid nanopore

4

5 nm

~15 nm

K+ Cl-K+K+

K+ K+

K+

K+K+

K+ Cl-

Cl-Cl-Cl-

Cl-

Cl-

Ea→~20 nm

b

10 nm10 nm

~20 nm

1 M KCl

~5 nm

Advantage: 1. better pore size control 2. modification around the pore

Disadvantage: 1. leakage 2. structurally more dynamic

5

Content

Introduction

Results:

(1) DNA origami expands under electric field

(2) [Mg2+] dependence of conductivity

(3) Voltage dependence of conductivity

Summary

Build a DNA origami system

6

(2) Introduce Mg2+ ion to neutralize the DNA

(3) Add water by VMD solvate plugin

(4) Add 1MKCl by VMD autoionize plugin

(1) Design the origami form caDNAno, make pdb file by cadnano2pdb[1], and make

scaffold periodicX Y

Z

[1]Yoo, Jejoong, and Aleksei Aksimentiev. "In situ structure and dynamics of DNA origami determined through molecular dynamics simulations." Proc Natl Acad Sci USA 110, no. 50 (2013): 20099-104. DOI: 10.1073/pnas.1316521110

7

Ion movement under electric field

E

K+ Cl-

Orchard: K+; Cyan: Cl-; Pink:Mg2+

D*

(c)(b)(a)

E

D

8

Nano accordion

HX2* HC2 SQ2

D*

(c)(b)(a)

E

D

9

Content

Introduction

Results:

(1) DNA origami expands under electric field

(2) [Mg2+] dependence of conductivity

(3) Voltage dependence of conductivity

Summary

10

0mM [Mg2+] 131mM [Mg2+] 246mM [Mg2+]

526453655883Area(angstrom2) =

[Mg2+] affects Conductivity by changing area

11

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Content

Introduction

Results:

(1) DNA origami expands under electric field

(2) [Mg2+] dependence of conductivity

(3) Voltage dependence of conductivity

Summary

13

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16

Content

Introduction

Results:

(1) DNA origami expands under electric field

(2) [Mg2+] dependence of conductivity

(3) Voltage dependence of conductivity

Summary

17

Summary

(1) DNA origami would expand under electric field. !(2) The conductivity of DNA origami would be affected by ion concentration, voltage and other factors.