Biotechnology and Bioprocess Engineering 19: 1-7 (2014)

DOI 10.1007/

Predictive Evaluation for the Preparation of a Synthetic Y-shaped

DNA Nanostructure

Kyung Soo Park, Seung-Won Shin, Jin-Ha Choi, Byung-Keun Oh, Jeong-Woo Choi, and Soong Ho Um

Received: / Revised: / Accepted:

© The Korean Society for Biotechnology and Bioengineering and Springer 2014

Abstract With the advent of deoxyribonucleic acid

(DNA) nanotechnology, the Y-shaped DNA nanostructure

(Y-DNA) as a basic block was first created. Due to their

characteristic selectivity and specificity, Y-DNA-based

materials have been utilized in a variety of scientific fields

including multiplexed nanobarcoding. Basically, the tripod

DNA nanostructure was prepared by simple hybridization

of three different single stranded DNA (ssDNA). Before

the synthetic process, the optical densities (OD) of the

three ssDNAs were measured to accurately estimate the

concentration. Through repeated temperature fluctuations,

three ssDNAs were hybridized into a Y-shaped block with

both a central junction and three blunt ended arms. After

the reaction, the ODs of the synthesized DNA products

were measured and compared with the theoretical OD

values calculated by a MATLAB program (‘matrix laboratory’)

with different molar concentrations and volumes to predict

the presence of Y-DNA. Simultaneously, the product was

analyzed by agarose gel electrophoresis to confirm the Y-

DNA structure. The measured ODs of the solutions with

confirmed Y-DNA structures were close to the theoretical

maximum OD values. This article provides means to help

understand and prepare Y-DNA by performing OD

measurements. It is highly expected that this guide will be

an excellent starting point for structural DNA nanotechnology.

Keywords: DNA nanotechnology, optical density, MATLAB

program, gel electrophoresis

1. Introduction

Along with the fast development of nanobiotechnology,

DNA has been used as an infra-structural block for a

variety of supramolecular nanostructures with conformational

flexibility at the nano-scale level and molecular specific

recognitions in predesigned manners [1-4]. For instance,

DNA origami [5] has been further applied to DNA tubes

[6,7], triangles [8], dimensional structures [9-11], and DNA

junctions [12-15]. In particular, Y-shaped DNA (Y-DNA)

junctions as a basic unit have been highlighted. Moreover,

by modifying the overhang sequences of each arm of Y-

DNA, the Y-DNAs ligate together in a controlled manner

resulting in a tree-shaped DNA supramolecular nanostructure

referred to as dendrimer-like DNA [16]. Furthermore, each

end in the DNA nanostructure may be functionalized with

various color codes in order to realize anisotropic built-in

and multiplexed barcodes [17-20].

To prepare such DNA blocks, each ssDNA is first

quantitated by measuring its optical density (OD) at a

wavelength of 260 nm. The initial OD measurement of

ssDNA is directly associated with the final product efficiency

and cost-effectiveness. Y-DNA is made by simply annealing

three single stranded oligonucleotides whose sequences are

complimentary to each other so that under the given

optimal conditions, the three oligo-pieces form Y-DNA as

designed. To prevent the loss of oligonucleotides, it may be

beneficial to accurately assess the quantitation of each

Seung-Won Shin, Jin-Ha Choi, Soong Ho UmSchool of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Korea

Soong Ho Um*

SKKU Advanced Institute of Nanotechnology (SAINT), SungkyunkwanUniversity, Suwon 440-746, KoreaTel: +82-31-290-7348; Fax: +82-31-290-7272E-mail: sh.um@skku.edu

Kyung Soo Park, Byung-Keun Oh, Jeong-Woo ChoiDepartment of Chemical and Biomolecular Engineering, Sogang University,Seoul 121-742, Korea

Jeong-Woo ChoiGraduate School of Management Technology, Sogang University, Seoul121-742, Korea

RESEARCH PAPER

2 Biotechnology and Bioprocess Engineering 19: 000-000 (2014)

DNA molecule and predict the state of the expected Y-

DNA before completely forming the Y-DNA. In the same

sense, quantitative evaluations of the created Y-DNA are

required to analyze its state before further applications. As

a result, a variety of experimental parameters must be

cautiously investigated such as the total reaction volume,

concentration of the synthesis solution, and dilution steps

taken before the OD measurements. To assess these

parameters, the three main variables were varied as

follows: (1) reaction volumes of 30 and 100 µL (2) molar

concentrations of 0.00600 and 0.00900 mM, and (3) one

dilution and two consecutive dilutions and for further

investigation, 2 or 10 µL of the stock solution was mixed

with DNase-free water for dilution.

Empirically, the measured OD was close to the theoretical

maximum OD. For example, the theoretical minimum and

maximum OD values of the solution with a concentration

of 0.00600 mM were 0.290 and 0.439, respectively, where

the average measured OD after nine trials was 0.445. It is

speculated that better products were generated in this study

for advanced DNA technological applications.

In this article, we provide a guide to a proficient

experimental and theoretical approach for the preparation

of Y-DNA by introducing detailed experimental information

which results in more precise OD measurements by

evaluating influential experimental parameters. Thus, it will

enable researchers to easily reproduce Y-DNA and predict

the final as-designed products, which will help confirm if

the Y-DNA was successfully synthesized. In addition, this

guide will be quite helpful for DNA nanotechnology novices.

2. Materials and Methods

Three complementary oligonucleotides, TE buffer (10 mMTris

(pH 8.0), 1 mM ethylenediaminetetraacetic acid (EDTA),

50 mM NaCl, DNase-free water (DFW), micro-pipettes,

graduated pipette tips, microtubes, a thermal cycler

(Eppendorf), and latex gloves were used in the experiments.

For the UV absorbance measurements of the DNA molecules

at a specific wavelength, which is typically 260 nm for

nucleic acids, a UV-vis spectrophotometer (BioPhotometer

Plus 6132, Eppendorf) and cuvettes (UVette®, Eppendorf)

were used.

2.1. OD measurement of ssDNAs and synthesized Y-DNA

To measure the OD of nucleic acids, three different

oligonucleotides, Y1, Y2, and Y3, were first separately

dissolved thoroughly in TE buffer with volumes of 500 µL

(Fig. 1A). 10 µL of each sample was then taken and

serially diluted into DFW to a final volume of 100 µL.

Then, the concentration of each oligonucleotide solution

was calculated by measuring OD of the solution at a

wavelength of 260 nm using a UV-vis spectrophotometer

(Fig. 1B). In order to measure the OD of a solution, the

solvent used to dissolve the oligonucleotides has to be

measured first to obtain the background signal. This

background is then subtracted from the total signal of the

oligonucleotide solution leaving only the signal from the

solute (oligonucleotide). If the OD value is larger than 1,

the sample solution must be additionally diluted until the

OD becomes less than 1 and within the machine’s

readability. Once the ODs of the ssDNA are measured, the

actual molar concentration of each oligonucleotide can be

calculated using the following equation.

33 µg/ml × Absorbance260 nm × Dilution factor =

DNA concentration (µg/ml)

Then, to synthesize Y-DNA, three oligonucleotides were

mixed at an equal molar ratio in a 0.1 mL microcentrifuge

tube and DFW was added to the final volume, which was

determined depending on the experiment design. The

Fig. 1. Schematic drawing of the overall experiment: (A) preparation of three oligonucleotides (ssDNA), where Y1, Y2, and Y3 (Table 4)are represented by red, yellow, and blue, respectively, (B) quantitation of each oligonucleotide through spectrophotometry, (C) annealingof the three oligonucleotides into Y-DNA using a thermal cycler, and (D) Y-DNA represented as three ssDNAs twisted into Y-shapedblocks after thermal hybridization.

Predictive Evaluation for the Preparation of a Synthetic Y-shaped DNA Nanostructure 3

typical designed total volumes of the solutions were 30 and

100 µL, which were used to evaluate the effect of the total

volume during the Y-DNA synthesis. First, to prepare a

standard solution that can be compared with other solutions

used for the synthesis, a Y-DNA solution with a concentration

of 0.00600 mM (total volume of 100 µL) was designed

where around 3.5 to 4.7 µL of each oligonucleotide

component was mixed with DFW (Table 1).

Next, the mixture was located in a reaction tube and

thermally cycled as programmed (Fig. 1C). As shown in

Fig. 2, the thermal cycle consists of three steps: denaturation,

cooling, and annealing. For denaturation, the DNA solution

is heated up to 95°C and held for 2 min. Then, the cooling

step proceeds where the temperature is cooled to 65°C and

held for 5 min. The cooling step is necessary to maintain

the solution in a stable state after denaturation. Then, to

initiate the annealing of DNA, the temperature is further

cooled to 60°C and held for 5 min. After 5 min, the

temperature is decreased at a rate of 0.1°C per minute and

repeated 40 times to optimize the annealing conditions.

The resulting Y-DNA solution is referred to as the ‘standard’

solution for comparison to the other synthesis conditions

used for the Y-DNA creation. After annealing is completed,

the concentration of the product sample was evaluated

using a UV spectrophotometer (Fig. 1D) following the same

steps described above where the ODs of the oligonucleotides

were measured. The sample was diluted up to a 1/10 ratio

with 10 µL of the sample solution diluted into 90 µL of

DFW so that the concentration was within the measureable

range.

The synthesized Y-DNA was further evaluated by a gel

electrophoretic migration assay. In running 3% agarose gel,

each lane contains different kinds of oligonucleotides with

a single strand in the first lane, incomplete strand complexes

in the second lane, and completes Y-DNA in the last lane.

They have different mobilities in the gel because of their

different molecular weights. These results will be shown

later along with a detailed discussion.

2.2. Theoretical OD calculation to determine the annealing

efficiency

Regarding how efficiently Y-DNA was created, we compared

the experimental data with data obtained by theoretical

tools. Briefly, the best results correspond to a product

efficiency of 100% and a product efficiency of 0%

represents the worst results. The relative results were

obtained by comparing the experimental and theoretical

OD values. Here, it is noted that the theoretical OD was

calculated using a simple modeling program (MATLAB).

For the program codes, the molecular weight and molar

concentration of the final product solution were set as

“input codes”. In MATLAB, the calculation process was

conducted to obtain the theoretical ODs using the above

input variables (Fig. 3).

These input codes are multiplied by each other to give a

theoretical concentration (µg/µL) of the DNA solution.

Depending on the presence of ssDNA or dsDNA, the

theoretical concentrations are divided by 50 (for dsDNA)

or 33 (for ssDNA) considering the corresponding theoretical

ODs. The final outputs are the theoretical minimum OD

(100% anneals with complete double stranded products)

and maximum OD (only single stranded DNA exists and

there are no anneals) (see Fig. S1 of Supplementary

Information). Y-DNA is created and the structure is in the

form of double strands and therefore theoretically, it must

Table 1. Oligonucleotide volumes for Y-DNA synthesis

Component Molecular weight Concentration (µg/µL) Mass (µg) Volume (µL)

Y1 8,051.2 1.028 4.831 4.699

Y2 8,020.2 1.150 4.812 4.184

Y3 8,082.2 1.396 4.849 3.474

DFWb 87.643

Total 100bDNase-free water.

Fig. 2. Thermal cycle of the Y-DNA annealing procedure. First,the temperature was increased to 95°C for denaturation and thencooled to 65°C to stabilize the state of the DNA. Then, theannealing steps (steps 3 and 4) were carried out. Step 4 isconducted by repeating 40 cycles where a cycle lasts for 1 minduring which the temperature is decreased by 0.1°C.

4 Biotechnology and Bioprocess Engineering 19: 000-000 (2014)

exhibit theoretical minimum OD values. If there are no

reactions for Y-DNA formation, only three single stranded

oligonucleotides may exist in the solution and it must

demonstrate theoretical maximum OD values. However,

experimentally, the ODs values of the synthesized Y-DNA

were around the theoretical maximum values.

2.3. Evaluation of synthesis conditions

To evaluate the influence of the synthesis conditions on

the Y-DNA creation, several solutions synthesized under

different parameters including the reaction volume, solution

concentration, and dilution factors were investigated by

measuring the OD at specific wavelengths of 230, 260,

280, and 340 nm. In addition, for more accurate results, the

entire steps from the solution synthesis to the OD

measurement were carried out nine times and then averaged.

First, to evaluate the effect of the solution volume, a solution

with a molar concentration and volume of 0.00600 mM

and 30 µL, respectively, was made (Table 2A) and compared

with the aforementioned ‘standard’ solution. Secondly, the

influence of how the dilution steps are performed on the

resulting OD was investigated. A solution with a

concentration and total volume of 0.00600 mM and 100 µL,

respectively, was prepared. Its parameters were kept the

same as those of the ‘standard’ and only differed in terms

of the dilution steps taken before measuring the OD. The

sample was diluted by a 1/100 ratio in two dilution steps,

each at a 1/10 ratio (Table 2B). Next, the evaluation of the

effect of the solution concentration to the Y-DNA creation

was conducted by making a solution with a molar

concentration and volume of 0.00900 mM and 100 µL,

respectively (Table 2C). As with the previous solutions,

its OD was measured and compared with that of the

‘standard’. Lastly, the dilution factors were evaluated. The

OD of a solution with a concentration and total volume

of 0.00667 mM and 30 µL, respectively, was measured

(Table 2D). During the OD measurement, the dilution was

carried out in two consecutive steps where the first step

diluted 2 µL of solution into 18 µL of DFW, resulting in

the intermediate solution at a 1/10 dilution ratio. Then,

10 µL of the intermediate solution was diluted into 90 µL

of DFW, resulting in a 1/100 diluted sample.

3. Results and Discussion

The full sequence information of the Y-DNA is given in

Table 3. The ‘standard’ solution synthesized at a molar

concentration of 0.00600 mM with a reaction volume of

100 µL was first evaluated. Before taking the OD measure-

ment, the Y-DNA formed was confirmed by a 3% agarose

gel electrophoretic assay (Fig. 4). Interestingly, the band

indicating the Y-DNA components composed of three

single strands was most slowly retarded because of the

Fig. 3. Algorithm of the MATLAB process used for the theoreticalOD calculations. The molar concentration (mM) and molecularweight are input codes which can be converted to the theoreticalconcentration (µg/µL). To hypothesize the condition for thetheoretical concentration calculation, the type of the DNA isdetermined whether to be single or double stranded and dependingon its type, the theoretical concentration is divided by 5 for singleor 3.3 for double stranded DNA yielding the theoretical minimumand maximum ODs, respectively.

Table 2. Optical densities of solutions obtained under different synthesis conditions

Standard (a) (b) (c) (d)

Concentration (mM) 0.00600 0.00600 0.00600 0.00900 0.00667

Total volume (µL) 100 30 100 100 30

OD (230 nm) 0.389 0.388 0.0343 0.557 0.06044

OD (260 nm) 0.445 0.433 0.0459 0.663 0.0569

OD (280 nm) 0.291 0.279 0.0342 0.279 0.0371

OD (340 nm) 0.000667 0.00122 0.00167 0.00122 0

STDV (260 nm) 0.00425 0.00372 0.00668 0.0119 0.00836

Dilution ratio 1/10 1/10 1/100 1/10 1/100

Dilution step (µL/µL) 10/90 10/90 (10/90)x2 10/90 2/18, 10/90

Predictive Evaluation for the Preparation of a Synthetic Y-shaped DNA Nanostructure 5

higher molecules weights (24,000 Da) relative to other

single strands (8,000 Da) and incomplete Y-DNAs consisting

of two single strands (16,000 Da). Compared with the

ladder DNA, it is evident that there are stepwise increments

of approximately 30 bp in the DNA nanostructures which

directly indicates the creation of Y-shaped DNA nano-

structures with three arms. Next, the OD of Y-DNA was

measured using a UV spectrophotometer (Table 2). The

OD260 nm obtained was 0.445 with a standard deviation of

0.004. It can be considered that the value is fairly reliable

when it is within the tenth digit of the OD and the standard

deviation is in the thousandth digit.

This experimental result was compared with the

theoretical OD calculated via MATLAB, in which we used

a molar concentration of 0.00600 mM and a molecular

weight of 24,153.6 as input codes. The calculated theoretical

minimum and maximum ODs were 0.290 and 0.439,

respectively, in which case the theoretical maximum OD is

similar to the experimentally obtained value of 0.445

(Table 4A). This phenomenon may be ascribed to the

characteristic topological features of Y-DNA [21]. It is

known that the topology or morphology of a molecule

affects its molecular optical properties.

Secondly, solution (a) in Table 2 whose reaction volume

was modified to 30 µL compared to the ‘standard’ was

investigated. For the MATLAB operation, because the

molar concentration and molecular weight are fixed to

0.00600 mM and 24,153.6, respectively, the theoretical OD

did not change from the previous case with minimum and

maximum OD values of 0.290 and 0.439, respectively. After

the nine trials of solution synthesis and OD measurements,

an average OD260 nm of 0.433 (Table 2A) was obtained,

which is again around the theoretical maximum OD

(0.439), which is slightly within the theoretical range. This

supports the above notion that light absorption of Y-DNA

with its unique morphology is somewhat different than that

of the common double stranded DNA. With only a subtle

decrease of the standard deviation compared to that of the

‘standard’ solution, a significant difference was not observed

when the total reaction volume was changed.

Next, the influence of how the dilution steps are performed

on the resulting OD was investigated. Solution (b) in Table 2

was prepared and its OD was measured. After carrying out

nine steps of solution synthesis and OD measurements, the

average was 0.0459 (Table 2B) which is similar to that of

the ‘standard’ solution (0.445) considering the one extra

dilution step, which requires multiplication by 10 for

Table 3. Oligonucleotide sequence

Strand Sequencesa

Y1 5'-Phos-TGGATCCGCATGACATTCGCCGTAAG-3'

Y2 5'-Phos-CTTACGGCGAATGACCGAATCAGCCT-3'

Y3 5'-Phos-AGGCTGATTCGGTTCATGCGGATCCA-3'aThe sequences were obtained from Li, Y.; Tseng, Y. D.; Kwon, S. Y.;D’espaux, L.; Bunch, J. S.; Mceuen, P. L.; Luo, D. Nat. Mater. 2004, 3:38-42.

Fig. 4. Stepwise confirmation of Y-DNA creation by agarose gelelectrophoresis: (1) 25 bp ladder DNA, (2) 26 bp singleoligonucleotide, (3) two 26 bp oligonucleotides partially annealedforming double oligonucleotides, and (4) triple oligonucleotidesannealed into Y-DNA forming a 78 bp DNA structure.

Table 4. Calculated theoretical optical density range

Theoretical OD Range

Molar concentration (mM) MW (g/mol) Theoretical concentration (µg/µL) Theoretical OD

(a) Minimum (ds) 0.00600 24,153.6 0.145 0.290

Maximum (ss) 0.0180 8,051.2 0.145 0.439

(b) Minimum (ds) 0.00900 24,153.6 0.217 0.435

Maximum (ss) 0.0270 8,051.2 0.217 0.659

(c) Minimum (ds) 0.00667 24,153.6 0.161 0.322

Maximum (ss) 0.0200 8,051.2 0.161 0.488

6 Biotechnology and Bioprocess Engineering 19: 000-000 (2014)

comparison. However, the standard deviation increased to

0.007. Considering the average OD is in the hundredth

digit, the standard deviation increased tenfold compared to

that of the ‘standard’ solution. It can be concluded that the

effect of taking extra dilution steps on the overall OD value

is not of significance but each trial may not be as precise

as that of when less dilution steps are taken considering the

increased standard deviation. Once again, similar to other

solutions, the measured OD was near the theoretical

maximum OD.

To evaluate the influence of the solution concentration

on Y-DNA creation, solution (c) in Table 2 was prepared.

It is usually expected that when the solution concentration

is higher, the solutes in the solution absorb more

transmitting light. The measured OD of 0.663 (Table 2C)

is higher compared to that of the previously mentioned

solutions, which are at lower concentrations. According to

the MATLAB program, the theoretical OD range was

calculated by applying a molar concentration of 0.00900 mM

and a solution volume of 100 µL as input codes resulting

in theoretical minimum and maximum OD values of 0.435

and 0.659, respectively (Table 4B). It was observed again

that the measured ODs were close to the theoretical

maximum ODs. However, the standard deviation almost

doubled compared to those of the ‘standard’ and previously

mentioned solutions. This may be due to the higher

concentration of the solution where only a small amount of

difference in every handling of the solution leads to a

higher deviation in the results.

Lastly, to test how the dilution factors influence the

solution OD, solution (d) was synthesized. It was preliminarily

concluded that if a lower amount of the original solution

was used compared to the above trials and diluted into

DFW, it induced less accurate results. Through the MATLAB

operation, the theoretical minimum and maximum OD values

of the 1/100 diluted solution with a molar concentration of

0.00667 mM and solute molecular weight of 24,153.6 were

calculated to be 0.0322 and 0.0488, respectively (Table 4C).

The measured OD result was 0.0569 (Table 2D), which is

not as close to the theoretical maximum OD as those of the

other solutions which underwent different dilution steps

where a larger volume (10 µL) of original stock solution

was diluted into DFW. This phenomenon may be ascribed

to the capillary phenomena occurring while handling a

much smaller volume using a micropipette.

It is concluded that the solution concentrations, volumes,

and dilutions during sample preparation do not have

significant effects on the OD values. Meanwhile, the

concentration is proportional to the OD and it was observed

that once the solution concentration was higher, the OD

deviated more from the overall OD obtained by averaging

many trials. While the samples were diluted, too small a

volume of the stock solution may cause substantial error in

the resulting OD. Empirically, we highly recommend that

at least 10 µL of a stock solution be used for dilution to

obtain reliable data based on the comparative and theoretical

ODs.

Interestingly, the DNA purity of the solutions also

influenced the preciseness of the measured OD. From the

ODs obtained at different wavelengths, the DNA purity of

the solutions can be deduced. The closer the OD340 nm is to

0 and OD260 nm/OD280 nm is to 1.8, the higher the DNA

purity. If OD260 nm/OD280 nm is lower than 1.8, some other

molecules that absorb light at a wavelength of 280 nm,

such as proteins or chemicals with phenols inside the

structure, exist within the solution. Lastly, OD260 nm/OD230

nm should be higher than OD260 nm/OD280 nm for a higher

DNA purity. Under different conditions, there were some

trials where the OD340 nm values were not zero (see Table S1

of Supplementary Information). Excluding those trials, the

average OD260 nm was recalculated which leads to a reduced

standard deviation. This means that if contamination is

reduced, more accurate results can be achieved.

4. Conclusion

The conditions which may influence the optical density of

a solution were assessed through comparisons to a standard

solution, in which Y-DNA was created and confirmed by

agarose gel electrophoresis. Other solutions that differed in

terms of any one of three synthesis parameters of

concentration, volume, and dilution steps were evaluated.

The volume was determined to not be a significant factor

when controlling a small amount of volume of less than

100 µL, whereas how the dilution steps were carried out

influenced the standard deviation of the obtained OD and

therefore, the preciseness of the results. The concentration

is proportional to the measured OD where an increased

concentration results in an increased OD. Also, changing

the concentration greatly affects the standard deviation

even when the reaction volume and dilution steps are fixed.

Additionally, if not contaminated by chemicals or biomaterials

other than DNA, more reliable data can be achieved which

can be reflected in a decrease of the standard deviation of

the average OD260 nm when trials with high values of OD340

nm are excluded from the calculation of the average. Also,

by comparing the measured OD260 nm with the theoretical

OD260 nm range, it could be concluded that solutions with Y-

DNA blocks successfully created had ODs around the

theoretical maximum OD. With the above result in mind,

knowing how the synthesis conditions affect the Y-DNA

creation, effective Y-DNA synthesis if possible even when

certain conditions need to be varied in different situations

Predictive Evaluation for the Preparation of a Synthetic Y-shaped DNA Nanostructure 7

simply by comparison with theoretical OD values obtained

using MATLAB rather than taking images after gel

electrophoresis, resulting in time and cost savings.

Acknowledgements

We thank Kyung-ho Nam and Hyun-ju Kim, both at the

School of Chemical Engineering of Sungkyunkwan

University, for their valuable advice and experimental

assistance. This work was supported by a grant from the

Korea Health Technology R&D Project of the Ministry of

Health & Welfare of the Republic of Korea (Grant No.

A110552) and by Basic Science Research Program through

the National Research Foundation of Korea (NRF) funded

by the Ministry of Education, Science and Technology

(2013R1A1A2016781).

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