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Effects of structural flexibility on the kinetics of DNA Y-junction assembly and gelation Lin Niu, Xuyan Yang, Wei Pan, Tao Zhou, Dongsheng Liu, Chengde Mao, and Dehai Liang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03299 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 12, 2016

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Effects of structural flexibility on the kinetics of DNA Y-junction assembly and gelation Lin Niu1, Xuyan Yang1, Wei Pan 1, Tao Zhou2, Dongsheng Liu2, Chengde Mao3*, Dehai Liang1* 1

Beijing National Laboratory for Molecular Sciences, Department of Polymer Science and

Engineering and the Key Laboratory of Polymer Chemistry and Physics of the Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China 2

Key Laboratory of Organic Optoelectronics & Molecular Engineering of the Ministry of

Education, Department of Chemistry, Tsinghua University, Beijing 100084, China 3

Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA

Corresponding Author

*Dehai Liang, Tel: +86-10-62756170, Fax: 86-10-62751708, Email: [email protected]

*Chenge Mao, [email protected]

ACS Paragon Plus Environment

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ABSTRACT. The kinetics of DNA assembly is determined not only by temperature, but also by the flexibility of the DNA tiles. In this work, the flexibility effect was studied with a model system of Y-junctions, which contain single-stranded thymine (T) loops at the center. It was demonstrated that the incorporation of a loop with only one thymine prominently improved the assembly rate and tuned the final structure of the assembly, while the incorporation of a loop of two thymines exhibited an opposite effect. These observations could be explained by the conformation adjustment rate and the inter-motif binding strength. Increasing DNA concentration hindered the conformational adjustment rate of DNA strands, leading to the formation of hydrogels in which the network was connected by ribbons. Therefore, the gel can be treated as a metastable state during phase transition.


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Introduction DNA nanotechnology,1-3 initiated by Seeman and his coworkers, provides a platform to create nanostructures by using single-stranded DNA (ssDNA) with designed sequence via selfassembly. In the past decade, well-controlled shapes, architectures, and materials, not only in 1D or 2D, but also in 3D, have been created by DNA nanotechnology.4-8 Since DNA is biocompatible and biodegradable, the structures and materials formed by DNA have been applied in the fields of nanomedicine, tissue engineering, biotechnology, and even nanomotors.5,6,9-13 Even though the progress on DNA structures and materials is rapid, the mechanism of DNA assembly has not reached an agreement. Schulman and Winfree attributed the DNA assembly to a nucleation and growth process. They demonstrated that the presence of seed molecules can remove the kinetic barrier of nucleation and facilitate the growth of the assembled structures.14-16 Ekani-Nkodo et al. studied the assembly of DNA nanotubes by fluorescence microscopy.17 They found that the DNA nanotube was elongated by both end-to-end attachment and overlap joining. Niemeyer and coworkers monitored the assembly of a 4*4 DNA tile by Förster resonance energy transfer (FRET) and observed two intensity decays, which were attributed to the formation of DNA tile and larger structure, respectively.18 Sobey et al. suggested that even the assembly of simple DNA tile involved multi-strand interactions that leads to the mismatched kinetic-trapped DNA structures.19 J. P. J. Sobczak et al. demonstrated that hundreds of DNA strands can cooperatively associate into complex nanoscale objects within minutes at constant temperature.4 Using in situ thermally controlled atomic force microscopy, Song et al. found that the integrity of the DNA origami is highly dependent on the annealing procedures.20 Our previous study


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indicated that the main energy barrier during DNA assembly came from the conformational adjustment of DNA strands, while the barriers in nucleation were negative.21 It has been reported that the persistence length of double-stranded DNA is about 50 nm22 (corresponds to 150 bp), while the persistence length of ssDNA is less than 2 nm 23 (corresponds to 1–3 bases). A typical DNA strand with 30-50 bases is in rigid rod conformation. But it turns into two random coils after being denatured. Therefore, the assembly of ssDNA into dsDNA via base pairing involves a coil-to-rod transition. The time and space range of transition depends on the sequence of DNA as well as the overall assembled structure. Since the rigid conformation possesses a much larger excluded volume than the coiled conformation of same length, the transition generates a large barrier during DNA assembly, especially at higher concentrations. Any factors enhancing this transition should facilitate DNA assembly. To test this hypothesis, we designed a Y-shape DNA tile (Scheme 1), which is able to form hydrogel via self-assembly. Each Y-junction contains three DNA strands. The sequences of the strands are listed in Scheme 1. 11-base-long segments 1 (blue), 2 (green), and 3 (red) are complementary to the segments 1’ (blue), 2’ (green), and 3’ (red), respectively. Segments a (brown), b (dark blue), and c (black) are 6-base-long, self-complementary sticky ends. They are designed in different sequence to increase the assembly directivity and to decrease the chances of generating loops. More importantly, a short segment of thymine (one or two Ts) is introduced, separately, in the bending point of 1 and 3’, 1’ and 2, 2’ and 3, to tune the flexibility of the DNA Y-junctions, and thus to facilitate the formation of hydrogels. The Y-junctions are thus correspondingly named as 0T, 1T, 2T. The assembly kinetics of the DNA tiles and the mechanical properties of the formed hydrogels have been investigated by laser light scattering (LLS), Atomic Force Microscopy (AFM), and


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rheometer. The experimental results show that the central thymine segments exhibit profound effect on the assembly of the Y-junctions.

Scheme 1. Y-junction and its assembly into hydrogel. The sequence of each strand is also listed with n = 0, 1, or 2.

Experimental procedures Materials. DNA strands with purity more than 99% (purified by PAGE) were purchased from Invitrogen Corporation (Shanghai, China). Tris base and EDTA were purchased from SigmaAldrich Co., LTD. Acetic acid and magnesium acetate were purchased from Beijing Chemical Reagent Company. All the reagents were used as received. Milli-Q water was used in all the experiments. Preparation of DNA solution. The three component DNA strands (2.0 µM each) were dissolved in TAE/Mg2+ buffer (pH 8.0), which contained Tris base (40 mM), acetic acid (20 mM), EDTA (2.0 mM), and Mg(Ac)2 (12.5 mM). The DNA solution was filtered through a 0.45 µm PES filter (Sartorius stedim biotech corp., Germany) at about 60 oC and collected in a dustfree glass vial. The vial with DNA sample was heated at 95 oC for 10 minutes by a thermos-stage


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and then quickly quenched to the thermos-bath in the LLS setup, which was preset at designated temperature. The time point of quenching was set at time zero. The assembly process was then monitored by time-resolved laser light scattering at 30o. Laser light scattering. Static light scattering (SLS) and dynamic light scattering (DLS) experiments were conducted on a commercialized spectrometer (Brookhaven Inc, Holtsville, NY) equipped with a BI-200SM Goniometer and a BI-TurboCorr Digital Correlator. A He-Ne laser polarized at the vertical direction (R-30995, 633 nm, 17 mW, Newport, USA) is used as the light source. The time averaged excess scattered intensity at angle θ, also known as the Rayleigh ratio Rvv(q), is related to the mass concentration C, the weight-averaged molar mass Mw, the Zaveraged root mean square radius Rg, the second virial coefficient A2, and the scattering vector q = (4π n / λ ) sin(θ / 2) as24 KC 1  1 2 2 ≈  1 + Rg q  + 2 A 2 C Rvv (q) M w  3 

(1)

2 2 2 4 where K = 4π n (dn / dC) ( N A λ0 ) , with NA, n, (dn/dC) and λ0 being the Avogadro constant,

the refractive index of the solvent, the specific refractive index increment of the solution, and the wavelength of light in vacuum, respectively. For simplicity, the excess scattered intensity Iex (denoted as (Is-I0)/It, with Is, I0, and It, being the scattered intensity from the solution, the solvent, and

toluene,

respectively),

which

is

directly

related

to

Rayleigh

ratio

௡

asܴ௩௩ ሺqሻ = ‫ܫ‬௘௫ ሺ‫ݍ‬ሻܴ௩௩,௧௢௟௨௘௡௘ ሺ௡ ೞ೚೗ೡ೐೙೟ ሻଶ, is used to show the progress of assembly. ೟೚೗ೠ೐೙೐

For dynamic scattering,

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the intensity-intensity time autocorrelation function G(2)(t) is

measured in the self-beating mode. It is related to the normalized first order electric field time correlation function g(1)(t) as G (2) (t ) = I (0) I (t ) = A 1 + β g (1) (t )2 

(2)


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where A is the measured base line, β is a coherence factor, t is the delay time. The line width distribution G(Γ) is analyzed with a Laplace inversion program, CONTIN25, based on the following relation, g (1) ( t ) =

∫

∞ 0

G ( Γ ) e − Γt d Γ

(3)

The average line width, Γ, is calculated according to Γ = ∫ ΓG (Γ)d Γ

(4)

and the polydispersity of a peak is defined as PDI =

µ Γ

2

2

2

2

= ∫ ( Γ − Γ) G ( Γ)d Γ [ ∫ G ( Γ) Γd Γ]

(5)

From the average line width Γ, the average apparent diffusion coefficient Dapp can be calculated 2 as Dapp = Γ / q , which is then converted into the hydrodynamic radius Rh, app by using the Stokes-

Einstein equation:

Rh,app = kBT / 6πη Dapp

(6)

AFM experiment. AFM imaging was conducted on Nanoscope III (Veeco Instrument Inc.) equipped with a 110 µm scanner. The images were collected in air at the tapping mode using commercial silicon tip (FESP, Veeco Instrument Inc.). The resonant frequency of the cantilever was about 70 kHz. For sample preparation, 30 µL DNA solution was deposited onto freshly cleaved mica surface. After 5 seconds, the extra solution was removed by a filter paper. Absorption of the DNA sample on the mica surface was allowed for another 5 min. The surface was then rinsed by 30 µL Milli-Q water for three times. The sample was naturally dried for 1 day before the AFM measurement.


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UV/Vis spectroscopy. The melting and formation of the DNA nanostructure was monitored by measuring the absorption of DNA at 260 nm using a UV/Vis spectrophotometer (Varian Cary 100, Varian, USA) with a temperature-controlled water bath. Temperature was controlled within ±0.1 °C. The sample solution at the concentration for laser light scattering was loaded into a screw-top cuvette and sealed firmly. It ensured no bubble formation in the solution and no evaporation during the heating. The heating/cooling process was conducted from 95 to 5 °C at a rate of 1 °C/min. Rheological experiments The mechanical property of 1T gel at 1.5 mM was studied by a rheometer (MCR301, Anton Paar). The amplitude sweeping was conducted at 10 rad/s at varying temperatures. The frequency sweeping is conducted at 10 oC at 0.1% strain. Scanning electron microscopy (SEM) 1T gel at 1.5 mM was flash-frozen in liquid nitrogen. The sample was further freeze-dried for 48 h until all the water was sublimed. The surface of the sample was peeled off and the fresh inner surface was metal-coated with Au/Pd. The imaging was observed via field emission SEM (S-4800 Hitachi, Japan).

Results and discussion The melting temperature of the DNA tiles was determined by UV spectroscopy. Figure 1 compares the melting (heating) and the formation (cooling) curves of the three DNA tiles. Only one major transition at similar temperature is observed in both the heating and cooling curves for all the DNA samples. The melting point is about 62 °C, and the formation point is about 60 °C. The difference suggests that a hysteresis exists during the assembly of DNA tiles at the cooling


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rate of 1 °C/min. These melting and formation curves suggest that the introduction of one or two unmatched Ts does not prominently change the base-matching behavior of the DNA strands.

heating cooling

2T Diff Abs

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1T

0T

40

60

80

100

o Temperature( C)

Figure 1. Differential UV absorption curves for the three DNA assemblies at 2.0 µM. The kinetics of DNA assembly was monitored by time-resolved laser light scattering at the temperature range of 15- 40 °C with 5 °C increment. No assembly was observed at temperatures above 40 oC by laser light scattering. Figure 2 shows the time dependence of the excess scattered intensity of 0T at selected temperature. Other results are included as supporting information (Figure S1). According to Eq. 1, the excess scattered intensity (Iex) is related to concentration, dn/dC, and Rg. At fixed concentration, the excess intensity is directly related to the size and density of the particles. As shown in Figure 2A, the assembly of 0T at 20 oC exhibits an “S” curve with heavy intensity fluctuation at later stage, which is similar to the assembly behavior of other DNA samples21. This can be explained by a nucleation and growth mechanism as reported26 . However, the excess scattered intensity starts to decline when it reaches a maximum value at temperatures ≥ 25 oC (see also Figure S1). The overshooting in excess scattered intensity


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suggests that the initial assembly of DNA tiles is not necessarily at equilibrium state. Similar results are obtained for 1T and 2T (Figures S2 and S3). 200

A. 20 oC

B. 25 oC 150

Iex

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20

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0 0

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Time/min

Figure 2. Time dependence of the excess scattered intensity of 0T at selected temperatures. C=2.0 µM. To reveal the effect of unmatched thymine, we compared the excess scattered intensity curves of 0T, 1T, and 2T at the same temperatures. As shown in Figure 3, the assembly rate of 1T is the fastest at all the studied conditions. The overshooting of the excess scattered intensity also occurs earlier in 1T sample. These results suggest that the incorporation of one unmatched thymine facilitates the nucleation and growth of the DNA strands, as well as the chain adjustment at later stage. Figure 3 also shows that the difference between 1T and 0T or 2T is larger at limiting temperatures, such as 20 oC or 35 oC. At the temperatures (25 oC and 30 oC) where the assembly is easier and faster, as demonstrated by the short induction time and fast growth rate, the differences between 1T and 0T or 2T are small (Figure 3B and 3C). The behaviors of 0T and 2T also show interesting results. The assembly of 0T is faster than 2T at lower temperature, such as


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20 oC (Figure 3A), while an opposite trend is observed at higher temperatures, such as 35 oC (Figure 3D).

120

120

A. 20 oC

B. 25 oC

100

Iex

80 80

60 0T 1T 2T

40 0 120 0

10

20 t/min

100

30

40

C. 30 oC

40 20 0 70

0

10

20 t/min

60

30

40

D. 35 oC

50

80 Iex

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40

60

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Time/min

Figure 3. Smoothed excess scattered intensity curves of 0T, 1T, and 2T at selected temperatures. C=2.0 µM. Figure 4 compares the hydrodynamic radius of the assembled structures of 0T, 1T and 2T at fixed temperatures. The size of the structure assembled by 1T is larger than those of 0T and 2T at most of the studied conditions. As for 0T and 2T, the size of 0T is generally larger at lower temperature, while the size of 2T is larger at higher temperature. This is in agreement with the trend of the excess scattered intensity. The major difference between the scattered intensity curves and the size curves is that the size monotonously increases with time, no overshooting is observed even at elevated temperatures. At fixed concentrations, the excess scattered intensity is proportional to the molecular weight. The ratio of Iex/R3 can be used to evaluate the density of the particles. As shown in Figure 5, the density of the assembled structure monotonously decreases with time in all the studied conditions. The targeting structure, as indicated by Scheme 1, contains pores with a diameter of about 12 nm, much larger than the size of a DNA strand.


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The initial DNA assembly via complementary base matching cannot generate such big pores. Instead, the DNA strands try to stick to each other as close as possible since the ssDNA are in random coil conformation and the hydrogen bond is short ranged. As many DNA strands bind together, the purpose of the sequence is then realized, and the adjustment of the structure results in a lower density. Figure 5 also shows that the densities of the structures in most of the cases are in the order of 0T > 2T >1T, further demonstrating the importance of proper chain flexibility. Figures 3 to 5 show that the initially assembled structure, especially at elevated temperatures, is not at final state. In the other words, the DNA strands, although their sequences are fixed, are “blind” to know the structure they are designed to assemble. 1T sample takes shorter time to “realize” the targeting structures. 1400

1400

o

A. 20 C 0T 1T 1000 2T 800

Rh/nm

1200

1200 800

600

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400

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200

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1400 1200

0

10

o

B. 25 C

1000

0

Rh/nm

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20

30

o

C. 30 C

0 40 0 10 1400 o D. 35 C 1200

1000

1000

800

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600

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400

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200

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0

20

30

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30

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0 0

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20 Time/min

30

40

0

10

Time/min

Figure 4. Changes in hydrodynamic radius of 0T, 1T, and 2T at fixed temperature. C=2.0 µM.


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ρ∼I/R3

10-4

10-4

A. 20 oC

10-5

10-5

10-6

10-6 0T 1T 2T

10-7

-4

10

0

10

B. 25 oC

10-7

10-8

ρ∼I/R3

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10-8 30 40 10-4 0 C. 30 oC

10-5

10-5

10-6

10-6

10-7

10-7

10-8

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30 40 D. 35 oC

10-8 0

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Time/min

30

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Time/min

Figure 5. Density curves of 0T, 1T, and 2T at selected temperatures. C=2.0 µM. To further analyze the effect of unmatched thymines, we calculate the activation energy for assembly by Arrhenius Equation,

κ = Ae−E / RT a

(8)

with A, Ea, R, and T being a frequency factor, the activation energy, the universal gas constant, and temperature, respectively. As Liu et al. have discussed,27 the reaction rate κ is inversely proportional to the nucleation time t, which can be obtained from the induction time in Figure 3. Since Ea is obtained by transferring Eq.8 to logarithmic form, the reaction rate can be directly replaced by 1/t without affecting the value of energy barrier. Therefore,

ln(1/ t ) = ln A - Ea / RT

(9)

Figure 6 shows the ln(1/t) versus 1/RT curves for the three DNA tiles. Firstly, the curves are not linear in the studied temperature range. They can be divided into two stages, with the turning point at 33 oC, which is independent of the number of unmatched thymines. The turning point is


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in between the melting points of the sticky ends (about 20 oC) and the central block (about 60 o

C). Similar results are also obtained on other DNA systems,21 indicating that the assembly of

DNA tile is a cooperative process between the sticky ends and the central block.

Secondly, all

the obtained energy barriers are negative, suggesting that the reaction rate decreases with increasing temperature. At lower temperature, the absolute values of the energy barriers are small and similar (1T is slightly lower), close to the energy of one hydrogen bond. This suggests that the nucleation can be trigged by the formation of one hydrogen bond, which is consistent with the cooperative model.18 However, the energy barriers are quite different at higher temperatures. The absolute value for 1T is 110 kJ/mol, about half that of 0T or 2T, suggesting that 1T is much easier to assemble. Note that the energy barrier obtained from Arrhenius Equation is meaningful only in primary interaction. Since the assembly of DNA strands contains both forward assembly and backward dissociation process, the obtained energy barrier is an apparent value. However, the assembly of Y-junctions can be treated as a quasi- primary reaction at lower temperature (< 33oC) when the forward assembly is dominant. -4.5 -33 kJ/mol -5.0

-5.5 ln(1/τ0)

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-110 kJ/mol

-44 kJ/mol -37 kJ/mol

-6.0

-185 kJ/mol

-6.5 -202 kJ/mol 0T 1T 2T

-7.0

-7.5 3.9

4.0

4.0

4.1

4.1

-4

1/RT/10


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Figure 6. Calculation of the energy barrier for the assembly of 0T, 1T, and 2T. C=2.0 µM. To further reveal the difference during assembly, we investigated the morphology of the structures formed by 0T, 1T, and 2T by AFM. Each DNA sample is annealed from 95oC to 25oC for 48 hours to ensure proper assembly. Figure 7 compares the AFM images of structures formed by the three DNA tiles at two different scales. 0T, due to its high rigidity, is able to assembly into large loose structures on the mica surface (Panel A and D). The height of the structure is only 2 nm, the same as the diameter of a double helix. The structure assembled by 1T is quite different. As shown in Panels B and E, 1T forms a closed structure about 120 nm in diameter. The height is 10 nm, four times higher than that of the structure formed by 0T. This suggests that the incorporation of one unmatched thymine significantly changes the assembly pattern of DNA tiles, which is in agreement with the findings by Mao et al.7 However, the incorporation of two unmatched thymines seriously deteriorates the assembly. As shown in Panes C and F, only small size particles with the height of 2 nm are observed.

Figure 7. AFM images of the structures assembled by 0 T (A, D), 1 T (B, E), and 2 T (C, F). C=2.0 µM.


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The effect of unmatched thymines can be explained by using our previously reported mechanism: the assembly of DNA is controlled by the binding strength, the conformational adjustment rate of DNA strands, and the spreading rate of new strands on the growing structure.21 The binding strength can be defined as the average number of matched base pairs per chain. To assemble into designed structures, the DNA strands will adjust the conformation to undergo a coil-to-rod transition. The unmatched thymines show profound effect on both factors. The incorporation of one thymine does not prominently affect the binding strength of DNA strands, but greatly enhances the conformational adjustment rate, lowering the energy barrier. 1T sample, therefore, exhibits a faster assembly rate. The incorporation of two thymines also prominently increases the conformational adjustment rate of the DNA strands, but it seriously decreases the binding strength by lowering the chances of base matching. At lower temperatures, binding strength is dominant during the assembly. 0T exhibits a faster rate than 2T. While the chain adjustment plays a key role at higher temperature, the assembly rate of 2T is thus faster than 0T. The chain spreading rate is faster at early stage due to the existence of large amount of free DNA strands in the solution. The assembly at this stage is not at equilibrium state since the DNA strands are “blind”. As the amount of free DNA strands decreases, the chain spreading rate also drops. When this rate is lower than the conformation adjustment rate, the assembled structure undergoes a conformation transition to correct the ill assembly generated at early stage. This is demonstrated as an overshooting of excess scattered intensity. Clearly, the conformational adjustment of DNA strand is the path to the structure at equilibrium state. If the adjustment of DNA stands is hindered during assembly, the structure will be kinetically trapped. One practical approach to hinder the conformation adjustment is to


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increase the concentration of DNA strands to confine the coiled structure in a limited space. Multi-stands interaction, which is greatly enhanced at elevated concentration, will create extra barrier for the conformation adjustment. To test this hypothesis, we studied the assembly of 1T by increasing the concentration by three orders to1.5 mM. Figure 8 shows the time dependence of the scattered intensity of 1T at 1.5 mM at fixed temperatures. The excess scattered intensity sharply increases at very beginning. No induction period is observed. The intensity then reaches a plateau and exhibits fluctuations. Not only the excess scattered intensity, but also the amplitude of the fluctuations increases with temperatures. As pointed out by Shibayama,26 such a periodical fluctuation indicates the formation of gels. This is understandable since the Y-junctions have three sticky ends and they have the capacity to cross link into network at elevated concentrations. Figure 8 also shows that no overshooting of the excess scattered intensity is observed, indicating that the DNA strands are confined in a limited space, and long-range conformation adjustment is hindered. A. 25 oC

Iex/It

20 15 10 5 0

Iex/It

o 20 B. 30 C

15 10 5 0 C. 35 oC

20 Iex/It

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Figure 8. Time dependence of the excess scattered intensity of 1T at 1.5 mM. Scattering angle 90o. To confirm that the conformation adjustment plays a key role in DNA assembly, we prepared 1T DNA gel by annealing method and studied its mechanical property by rheometer. Figure 9A shows the amplitude sweeping at different temperatures. The storage modulus G’ at 10 rad/s increases with lowering temperature, while the linear viscoelastic region, in which G’ is constant, shows an opposite trend. For example, the maximum G’ is about 180 Pa at 4 oC, higher than that at 15 oC. However, the linear viscoelasticity region at 15 oC is about 1% strain, 10 times higher than that at 4 oC. This clearly demonstrates that the hydrogen bonding at lower temperature is stronger, while the conformation adjustment is facilitated at higher temperature. Figure 9B shows the frequency sweeping of 1T at 10 oC at 0.1% strain. G’ is larger than G” at the studied conditions, suggesting that the gel formed by 1T at 10 oC exhibits strong elastic behavior. However, G’ still exhibits a frequency dependence, and G” increases at lower frequency, implying that the gel structure formed by Y-junction contains dangling ends and is subject to flow with increasing time. Assuming that all the Y junctions assemble into network structure as designed, the storage modulus could be calculated by the equation for elasticity of a rubber,28 GT = vkBT, with v, kB, and T being the number density of elastically effective network strands, the Boltzmann constant, and the absolute temperature, respectively. Since one DNA strand contributes 1/3 network strand, GT = 1/3µkBT (µ is the number density of the DNA strands). Calculation shows that GT is 3.5 × 103 Pa at 4.5 mM at 10 oC, which is 3~6 times larger than the measured G’ value (from 500 Pa to 900 Pa depending on frequency) in Figure 9B. This means that more than 60% of the Y-


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junctions do not form effective cross-linking points as expected, suggesting that the DNA strands are kinetically trapped in certain state, which contains many loops and unpaired dangling strands. 250

4 oC

A

o

200

10 C

G'/Pa

o

15 C

150

20 oC o 25 C

100 50 0 0.01

0.1

1

strain/% 10000

B

G' G''

1000 G/Pa

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100

10 0.01

0.1

1

10

Frequency/Hz

Figure 9. (A)amplitude sweeping of 1T at 1.5 mM at different temperatures (Angular Frequency 10 rad/s), (B) frequency sweeping of 1T gel at 10 oC at 0.1% strain. SEM was then conducted to study the morphology of the DNA gel after freeze-dry. As shown in Figure 10, the overall structure (Figure 10A) is a network. The connection is achieved by ribbons instead of thin fibers. It is understandable since the Y-junctions are designed to form sheet-like structures with a pore size about 12 nm, which is not detected by SEM. The porous structure in Figure 10 suggests that the gel can be treated as a metastable state during phase separation.

This is similar as the physical gelation of polymers. Keller29 pointed out that

crystallization was often regarded as the principle source of polymer gelation: the fibrous crystals, which resulted in polymer network at later stage, arose from the crystallization of


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polymer chains where folding was difficult or outright impossible. The gelation of Y-junctions in the current study could follow a similar approach except that the limitation of chain relaxation hinders the coil-to-rod transformation, and thus traps the DNA assembly in gel state.

Figure 10. SEM images of the 1T gel at two different magnifications. The concentration of DNA is 1.5 mM. Conclusions The flexible ss-DNA chains have to undergo a random coil-to-define structure transition during the process of assembling into the predesigned structures. The conformation adjustment rate of the DNA strand is thus one of the key parameters determining the kinetics of DNA assembly. Our study demonstrates that the incorporation of one thymine in Y-junction to increase the flexibility of the strand is able to prominently improve the assembly rate, as well as the final assembled structure. Kinetic study on the DNA assembly at low concentrations reveals that the DNA strands cannot predict the final structure they are designed to assemble. But they can “feel” it by base matching during the multi-chain assembly process. Under the conditions


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that the conformation adjustment is hindered, the assembly will be trapped in certain stage. The hydrogel formed by Y-junctions at higher concentrations contains a network of ribbons, which can be treated as a metastable state during the liquid-to-solid transformation. The gelation via kinetic path not only helps to understand the assembly of DNA strands, but also shed light on the phase transition of DNA, and probably polymers and small molecules.

ASSOCIATED CONTENT Supporting Information. Time dependence of the excess scattered intensity of DNA samples at selected temperatures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Dehai Liang, Tel&Fax: +86-10-62756170, Email: [email protected]; Chengde Mao, [email protected]. ACKNOWLEDGMENT This work is partially supported by the National Basic Research Program of China (973 Program, 2012CB821500), and the National Natural Science Foundation of China (# 21174007). REFERENCES (1)

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