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12216 Chem. Commun., 2012, 48, 12216–12218 This journal is c The Royal Society of Chemistry 2012

Cite this: Chem. Commun., 2012, 48, 12216–12218

DNA cohesion through bubble–bubble recognitionwzHang Qian,

abJinwen Yu,

bPengfei Wang,

bQuan-Feng Dong*

aand Chengde Mao*

b

Received 29th September 2012, Accepted 30th October 2012

DOI: 10.1039/c2cc37106e

This communication reports a novel intermolecular interaction

for structural DNA nanotechnology.

Successful, tile-based self-assembly of DNA nanostructures

depends on well-defined nanomotifs and predictable inter-tile

interactions.1–6 Great efforts have been devoted to the develop-

ment of DNA nanomotifs,1–25 but little progress has been made

for novel inter-tile interactions besides sticky-end cohesion.

Three noticeable exceptions are Paranemic Crossover (PX)

interaction,26 T-junctions,27 and edge-sharing.28 Here we report

a novel inter-tile interaction: bubble–bubble association that

does not require any free DNA ends.

Fig. 1 illustrates the reported intermolecular DNA bubble–

bubble cohesion, which results in a parallel double crossover

(P-DX) structure.29 P-DX is generally regarded as unstable and

its component strands often form a mixture of alternative DNA

complexes with different molecular weights. The ill-behaviour of

P-DX is attributed to strong electrostatic repulsion because of the

direct juxtaposition between the negatively charged backbones of

its two component DNA duplexes (Fig. 1e).29,30 We hypothesize

that such electrostatic repulsion could be avoided if two arms

beyond the crossover points are short enough (less than a half

helical turn). To test this hypothesis, we have designed two bubble-

containing DNA duplexes (Fig. 1a). One is colored red (R) and the

other green (G). The bubbles are six bases long. At one side of the

bubble, each molecule has a short hairpin containing a 3-base pair

(bp)-long stem and a four-base-long (T4) single-stranded loop. The

two bubbles have complementary sequences and are potentially

able to recognize and bind with each other (Fig. 1a and b).

Furthermore, the two bubbled DNA duplexes (Fig. 1c) can be

fused together into one fused molecule (F) that contains two

complementary bubbles. Multiple copies of the F molecules will

be able to self-assemble into linear structures (Fig. 1d).

We first studied the proposed bubble–bubble cohesion by

native polyacrylamide gel electrophoresis (PAGE, Fig. 2). Under

common DNA self-assembly conditions (with 10 mM Mg2+), the two individual bubbled DNA duplex molecules (R and G)

formed a heterodimer (R/G), whose mobility appeared to be

similar to that of the fused molecule (F) as the R/G heterodimer

and the F molecule had exactly the same molecular weight. The

F molecule self-organized into a series of linear structures:

monomer, dimer, trimer, up to decamer; corresponding to the

bands in the right lane in the gel image. It was noticed that not all

of the bubble molecules formed a dimer presumably due to the

electrostatic repulsion either between or in the two component

DNA duplexes. To reduce the electrostatic interactions, we

increased the Mg2+ concentration to 50 mM; under such a

Fig. 1 Bubble–bubble cohesion. (a) Two bubbled DNA duplexes

(red, R and green, G) interact with each other by complementary

Watson–Crick basepairing as exemplified by one set of DNA

sequences. (b) Conformation of the bubble–bubble interaction. (c)

Fusion of the two bubbled duplexes (R and G) into one fused molecule

(F) that contains two complementary bubbles. (d) The F molecules can

self-organize into long linear structures through bubble–bubble

cohesion. Alternating colors are used for the F molecules in the

assembled, linear structure to make each individual F molecule clear.

(e) A traditional parallel double crossover (P-DX) structure. Solid

yellow circles indicate locations of direct juxtaposition of negatively

charged backbones between the two component DNA duplexes.

a State Key Laboratory for Physical Chemistry of Solid Surfaces andDepartment of Chemistry, College of Chemistry and ChemicalEngineering, Xiamen University, Xiamen, Fujian 361005, China.E-mail: qfdong@xmu.edu.cn

bDepartment of Chemistry, Purdue University, West Lafayette,Indiana 47907, USA. E-mail: mao@purdue.edu;Fax: +1-765-494-0239; Tel: +1-765-494-4098

w This article is part of the ‘Nucleic acids: new life, new materials’ webthemed issue.z Electronic supplementary information (ESI) available: Experimentalprocedures. See DOI: 10.1039/c2cc37106e

ChemComm Dynamic Article Links

www.rsc.org/chemcomm COMMUNICATION

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This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 12216–12218 12217

condition, the R/G heterodimer yield greatly increased (to almost

100%). The F molecule also formed polymers far larger than the

decamer that migrated slower than the 1500 bp DNA duplex.

Some of them were so large that they could not even penetrate

into the gel matrix.

Atomic force microscopy (AFM) imaging confirmed the F

molecule self-association (Fig. 3). After assembly, the F

polymers were deposited onto the mica surface and visualized

by AFM. Linear structures were clearly observed; confirming

that F molecules could self-organize into linear polymers.

We further studied the bubble–bubble cohesion by thermal

denaturation (Fig. 4). Each individual bubbled duplex (R or G)

had only one thermal transition (between 60–80 1C),

corresponding to the melting of the basepairing within the

individual component DNAmolecules. When the two bubbled

DNA duplexes (R + G) were mixed together, a new thermal

transition emerged (at B45 1C) in addition to the original

thermal transition. The new transition was due to the dissociation

of the bubble–bubble interaction. Not surprisingly, the F

molecule itself exhibited both transitions corresponding to the

denaturation of the (inter-molecular) bubble–bubble interaction

and the (intra-molecular) residue basepairing.

Compared to P-DX, the bubble–bubble cohesion structure

with two short arms is stable. To estimate the impact of the

arm length on the stability of such interaction (Fig. 5), we

designed a series of bubble-containing DNA molecules with

different stem length (2–5 bps). The DNA sequences in the

bubble region are self-complementary, thus the DNA

molecules can self-dimerize through bubble–bubble cohesion.

In our hypothesis, when the stems are short (3 bp-long), the

Fig. 2 Native polyacrylamide gel electrophoretic (PAGE) analysis of

DNA bubble–bubble cohesion at different Mg2+ concentrations (10 or

50 mM). The DNA sample compositions are indicated above the gel

images and the identity of each band is indicated at the sides of the gel

images. The size markers contain a series of DNA duplexes.

Fig. 3 Atomic force microscopy (AFM) image of the linear polymers

assembled from the F molecule in the presence of 50 mM Mg2+. (a)

A large view field and (b) its zoom-in view. Height scale bar is shown

on the right.

Fig. 4 Thermal denaturation analysis of DNA bubble–bubble

cohesion. The transitions of intra- and inter-molecular basepairing

are indicated. Note that the four curves are manually shifted vertically

for clarity.

Fig. 5 Impact of the stem length. (a) DNA bubble molecules

that contain bubbles with self-complementary sequences can form a

homodimer through bubble–bubble cohesion. The stem sequences

(2–5 basepairs, bps) of study are colored red. The DNA molecules

are named with the stem length of concern. Solid yellow circles

indicate the locations of potential electrostatic repulsion between the

backbones. (b) Thermal denaturation profiles of DNA molecules with

different stem lengths. The melting temperature (Tm) of the

bubble–bubble interaction is indicated with the same color as the

thermal profile for each molecule.

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12218 Chem. Commun., 2012, 48, 12216–12218 This journal is c The Royal Society of Chemistry 2012

electrostatic repulsion between the top duplex and the bottom

duplex will be small; hence, the overall structure will be stable

and has a high melting temperature (Tm). As the stem length

increases (4 or 5 bp long), the backbones of the two duplexes

will directly juxtapose (highlighted by solid yellow circles) to each

other, resulting in strong electrostatic repulsion and destabilizing

the overall structure. Thus, the overall structure will have lower

Tm values. However, when the stem is too short (2 bp long), the

duplex stem itself becomes unstable; no bubble–bubble inter-

action would be expected. The thermal denaturation experiment

is consistent with this hypothesis (Fig. 5b).

In conclusion, we have introduced a novel interaction

between DNA nanomotifs: bubble–bubble cohesion, which

has been demonstrated by thermal denaturation and PAGE.

This interaction does not require free single-stranded ends

(sticky ends). It has a potential advantage over sticky-ends

association because it could be compatible with denaturing gel

purification of building motifs.26 Such purification might be

important for large, complicated motifs.

This work was supported by the Office of Naval Research.

H.Q. would like to thank China Scholarship Council for

financial support.

Notes and references

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