DNA Sequential Logic Gate Using Two-Ring DNA - ACS Applied

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DNA sequential logic gate using two-ring DNA Cheng Zhang, Linjing Shen, Chao Liang, Yafei Dong, Jing Yang, and Jin Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00847 • Publication Date (Web): 18 Mar 2016 Downloaded from http://pubs.acs.org on March 24, 2016

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ACS Applied Materials & Interfaces

DNA Sequential Logic Gate Using Two-Ring DNA Cheng Zhang,1* Linjing Shen,2 Chao Liang,3 Yafei Dong,2 Jing Yang3*and Jin Xu1* 1

Institute of Software, School of Electronics Engineering and Computer Science, Key Laboratory

of High Confidence Software Technologies, Ministry of Education, Peking University, Beijing 100871, China. Email:[email protected]; [email protected] 2

College of Life Science, Shannxi Normal University, Xi’an 710062, China

3

School of Control and Computer Engineering, North China Electric Power University, Beijing

102206, China. Email: [email protected] KEYWORDS DNA logic gate, sequential detection, interlocked structure, two-ring DNA, gold nanoparticle

ABSTRACT

Sequential DNA detection is a fundamental issue for elucidating the interactive relationships among complex gene systems. Here, a sequential logic DNA gate was achieved by utilizing the two-ring DNA structure, with the ability to recognize “before” and “after” triggering sequences of DNA signals. By taking advantage of a “loop-open” mechanism, separations of two-ring DNAs were controlled. Three triggering pathways with different sequential DNA treatments were distinguished by comparing fluorescent outputs. Programmed nanoparticle arrangement guided by “interlocked” two-ring DNA was also constructed to demonstrate the achievement of designed nanostrucutres. Such sequential logic DNA operation may guide future molecular sensors to monitor more complex gene network in biological systems.

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Introduction DNA is an ideal material for fabricating nanostructures and devices based on highly specific hybridizations. Recently, DNA self-assembly has attracted great interest in designing functional nano-systems, including controllable molecular nanomechanical devices, nucleic acid probing, and DNA computation.1-9 Particularly, in recent years, the development of DNA computing has also integrated with the related nano-engineering fields10-13 and has been applied to molecular detection,14,15 logic circuits,16-18 and nanodevices.19,

20

In fact, most of the well-established

previous DNA logic operating methods have been based on combinational logic, which is determined by the current inputs “0” or “1”.

21, 22

In other words, the outputs of DNA

combinational logic often depend on the combination of their DNA signal inputs, and have no “sequential memory” to detect triggering orders. However, during the course of natural gene expression, several genes may be transcribed cooperatively and sequentially in specific chronological sequences.23 Therefore, it is advantageous to develop a DNA sequential logic detection system that can not only monitor logic molecular triggers but can also recognize the different treatment sequences. Many operating methods are involved in DNA logic operations, such as DNAzyme recognition,24 DNA nanoparticle conjugates,25, 26 and DNA strand branch migration.27-30 One key feature of these DNA computing devices is that logic operations are mainly implemented using chemical methods such as hybridization and cleavage, lacking switchable and transformable mechanisms. In comparison with the previous chemical operations, it is interesting to introduce physical strategies into DNA logic operations. In practice, there have been numerous structural and kinetic studies of physical effects on switchable DNA structures, which provide additional operating methods for programming complex dynamic DNA configurations


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and detecting DNA signals.31,

32

Particularly, DNA ring systems with interlocked topologies,

separable motifs, and mobile bonding represent an attractive structure for molecular devices. 33-39 Previous studies have already demonstrated the feasibility and controllability of DNA ring molecules. However, most of the DNA rings in these studies used closed loops generated by T4 DNA ligase without any loop-open ability. In addition, the intermediate state, consisting of interlocked and unhybridized two-ring DNA, is an interesting dynamic structure that warrants further investigation. In this study, DNA sequential logic gate was achieved using a two-ring DNA structure. In contrast to previous logic circuits that only responded to the combinaitonal inputs, our logic detection can take into account the sequential treatments of DNA signals to recognize the chronological sequence of “before and after”. Here, three triggering pathways with different sequential treatments were distinguished by fluorescent outputs. Meanwhile, the configurations of two-ring DNA were regulated by using a “loop-open” mechanism. Finally, the programmed nanoparticle arrangement guided by “interlocked” two-ring DNA was also constructed. Experimental Section Materials. Gold nanoparticles (AuNPs) of 5, 10, and 15 nm diameters were purchased from Ted Pella; Bis(p-sulfonatophenyl) phenyl phosphine dipotassium salt dihydrate (BSPP) was purchased

from

Strem

Chemical;

and

HPLC-purified

disulfide-protected

thiolated

oligonucleotides were purchased from Sangon China. Native PAGE. To generate the DNA assembly scaffolds, the reactions were accomplished in an 80 µL 1× TAE buffer, using a DNA concentration of 2 µM and incubation of 2 hours at room temperature.


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Fluorescent signal detection. The fluorescent results were obtained using a fluorescent scanning spectrometer (Hitachi, F-2700) for HEX at 535-nm excitation and 556-nm emission. It should be noted that, in fluorescent detection, all the DNA catenane products were purified by direct cutting from the PAGE gel. Specifically, the cut and chopped gel was incubated with 500 µL 1× TAE/Mg2+ buffer with overnight agitation. Then, the sample was concentrated by Amicon Ultra 0.5 mL filters (10K). Later, the concentrated sample was diluted to 0.15 µM. In a typical reaction, the volume of the diluted products was 120 µL, and the final concentrations of DNA Ad and A1d were 1.5 times higher than those of the diluted products, with a volume of 5 µL. Phosphination of AuNPs. Solid powder BSPP powder was added to AuNPs solutions at a ratio of 25 mL to 12.5 mg. The mixed solution was stirred for 8 hours at room temperature. Then, powdered NaCl was added until the solution slowly changed from red to purple in color. The color changing solution was centrifuged at 5000–8000 rpm for 15–25 min to obtain AuNPs depositions. Finally, the AuNPs solution was re-diluted in 300 µL 1× BSPP solution (10× BSPP solution: 1.24 mg BSPP in 1 mL water). Preparation of AuNP/DNA Conjugates. In this study, the number of DNA strands attaching onto one particle was controlled precisely by controlling the ratio of DNA and AuNPs. To generate monovalent AuNP/DNA conjugates, thiolated DNA strands were incubated with freshly prepared AuNPs at a ratio of 1:1 or 1:2, in 0.5× TBE buffer with an NaCl concentration of 50 mM, for 4–6 hours at room temperature. With different diameters of AuNPs, concentrations of DNA were changed accordingly. For 5 nm AuNPs, the DNA concentration was about 0.8–1.2 µM. For 15 nm AuNPs, the DNA concentration was about 0.15–0.25 µM. Similarly, to generate multivalent AuNP/DNA conjugates, the DNA concentration was increased 30 times compared to


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that used in preparing monovalent conjugates. After the conjugations of thiolated DNA and AuNPs, agarose gel (2%) was used to extract the target conjugates.

Results and Discussion Here, a switchable DNA two-ring was used as a basic unit to implement the sequential logic operations, with regulated programmable separations of the two DNA rings. Figure 1 depicts the method for synthesizing the two-ring DNA C. First, to assemble the two-ring DNA, two singlestranded DNA (ssDNA) molecules A and B were hybridized to form A+B with a 12-mer complementary hybridizing region (12-merR). Then, in the presence of ssDNA A1 and B1, structure A+B hybridized with them to yield the two-ring DNA. Since the 12-merR is slightly longer than the length of one turn of DNA double helix (10.5 bp), this hybridization could generate two products: interlocked product C1 and uninterlocked product C2 (without the genuine interlocked structure, Scheme S1). Notably, when forming the two-ring DNA product C, the products C1 and C2 could be generated at the same time and were not separated. Nevertheless, product C2 could be generated by individually constructing DNA rings A+A1 and B+B1, and then mixing the preformed rings together (method 2 in Scheme S1).

Figure 1. (a) The design of forming the two-ring DNA products C. (b) PAGE gel results for generating two ring products.

Notably, a specific toehold region (labeled with yellew in Figure 1a) was designed in the middle of strand A to recognize the ssDNA input Ad. As hybridization between Ad and strand A (an 18 bp hybridizing region) was more energetically favorable than that between A and B (a 12


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bp hybridizing region), the addition of Ad would result in the disassembly between strands A and B. For the interlocked product C1, however, adding Ad alone could not lead to separation between the two DNA rings due to the physical connection of the “interlocked” topology of the two-ring (intermediate states S1, Figure 2a). For the uninterlocked product C2, addition of Ad alone was enough to separate the two rings (State S2).. In addition, to implement “loop-open” mechanism, another specific toehold (labeled with green in Figure 1a) at the 5’ end of strand A1 was also designated to open the DNA ring A+A1. Interestingly, only by adding the ssDNA strands Ad and A1d, both structures of C1 and C2 were broken, and the two DNA rings would be separated.

Figure 2. (a) The principle of separating the DNA two rings by DNA strand displacement. (b and c) The PAGE gel of strand displacing operations (DNA concentration was 2 μM). The PAGE gel using fluorophore modified DNA: B-HEX and Ad-FAM, modified with fluorophores HEX and FAM, respectively.

To achieve the sequential logic operations, two-ring DNA was firstly tested by PAGE gel as shown in Figure 2 b and c. When no input signal was added, the gel band of the two-ring product C, as shown in Figure 2b lane 3, indicated that its structural integrity was maintained. Consistent with our design, by adding Ad into product C, product C1 was transformed into an intermediate state product S1 as shown in Figure 2b lane 5 (with the same migration speed as product C in lane 3). In this case, product C2 was completely separated into two single DNA rings (two gel bands indicated by red and blue arrows in lane 5). Likewise, by adding input A1d into product C, the ring A+A1 was opened, and the two DNA rings could not be separated from each other due


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to the hybridization between them (Figure S2c, lane 9). Finally, when inputs A1d and Ad were added together, both products C1 and C2 were separated completely, without the presence of the gel band of initial product C (lane 7). As a control, uninterlocked two-ring DNA product C2 was also investigated by triggering with inputs Ad and A1d (Figure 2b). It is remarkable that the yield of product C2 was much lower than that of product C1, and a large number of single rings of A+A1 and B+B1 were left unconnected (compare the gel band intensities in Figure 2b lanes 3 and 4, and Figure S2b lanes 7 and 8). The possible reason may be that the pre-hybridized double-stranded regions of rings, along with the stiffness of the loop structures, greatly prevented flexible turning during binding of the two rings. In the presence of Ad, the gel band of product C2 disappeared almost completely, as shown in Figure 2b lane 6 (also seen in Figure S2b lanes 4 and 6). These results clearly indicate that, the small topological difference between products C1 and C2 can lead to distinct differences in configurations of the two-ring DNA during strand displacement. In addition, in order to better visualize the PAGE results, strands B and Ad were fluorescently modified with HEX (orange) and FAM (green), respectively, as shown in Figure 2c.

Figure 3. (a) The principle of increments of fluorescent signals. (b) Logic circuit diagram for sequential detection. (c) Fluorescent results of DNA two-rings in three triggering pathways. The values in the graph was obtained from three repeated detections at one time points (The time interval was 3 min). (d) The normalized results of sequential logic gate in Table 1. The average values in the graph was obtained from last three detections during the each stationary phase (the threshold was set to 1.0). (NOTE: The fluorescent results were normalized by FI=△F/F0, where


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F0 is the fluorescent intensity in initial state without adding any input strands and △F is the increment of the fluorescent intensity.

In the detections of sequential logic results, a sensitive fluorophore-quencher method was used. As depicted in Figure 3a, quencher BHQ and fluorophore HEX were modified in the middle of the DNA strands A and B, respectively. When product C was generated via hybridization of 12merR, fluorophore HEX on strand A was quenched, due to the close distance between the quencher and fluorophore. First, treating product C with the input Ad would directly result in strand displacement in the 12-merR domain. Then, the two rings of product C2 would be directly separated and a fluorescent increment (1st FI) would be observed accordingly. Meanwhile, for product C1, although the two rings were interlocked without hybridization, the fluorophore was still quenched by BHQ due to the relative vicinity. Second, when treating product C with two inputs Ad and A1d in succession, the fluorescent increment (1st FI) would be observed at first, and then another fluorescent increment (2nd FI) would be produced after the addition of A1d, resulting from the complete separation of interlocked product C1 in state S1. Here, three triggering paths with different addition orders were used to realize sequential logic operations (Figure 3c), as pathway 1: Ad→A1d (blue line), pathway 2: Ad→TAE buffer (green line), and pathway 3: A1d→Ad (red line). In pathway 1, the input Ad was first added to product C, and a significant fluorescent increment (1st FI) was produced. Apparently, generation of the 1st FI was mainly caused by strand displacement of product C2, which was a non-interlocked structure, and the two DNA rings could be easily separated. After the first addition, the intermediate state S1 was generated with the physical interlocked connections (Figure 3a). Then, in the presence of the second input A1d, another significant fluorescent increment (2nd FI) was


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detected accordingly. Here, the fluorescent increment 2nd FI was attributed to the complete disassembly of fluorophore and quencher, caused by the open ring mechanism. On the other hand, it should be noted that, although the fluorophore and quencher on the two rings were not tightly connected in the intermediate state, the quenching effect was still enough to restrain the fluorescent increments (Figure 3a). In pathway 2, the first addition of Ad similarly triggered the 1st FI as in pathway 1. While the second addition of the TAE buffer was not able to induce any significant fluorescent increment. In contrast, for pathway 3, the first addition of A1d was not able to induce any significant fluorescent increments. Only after adding Ad, could a quickly increasing fluorescent signal be detected (Figure 3c, red line), suggesting the complete dissociation between the two rings. The detection results are summarized in Table 1 (Figure 2c). In particular, although pathway 1 and 3 had the same inputs of Ad and A1d, it was still easy to distinguish them depending on specific fluorescent results. On the other hand, to demonstrate whether the specific 2nd FI was indeed induced by the intermediate state S1, two incomplete structural assembling molecules were utilized as products P1 (green line) and P2 (pink line) in Figure 4. The triggering process was implemented in two steps: first adding Ad, and then A1d. As expected, when investigating product C, both the 1st FI and 2nd FI were detected accordingly. However, when investigating P1 and P2, the first addition of Ad induced a significant 1st FI, whereas the second addition of A1d was not able to trigger any 2nd FI. This observation provided further evidence that without the physical connections from interlocked structures, the 2nd FI could not be produced.


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Figure 4. Fluorescent results by using incomplete structures P1 and P2. (The time interval was 3 min).

To further investigate the structures of product C, a programmed AuNP/gold nanoparticle assembly system was established. Gel results in Figure 5a show the assembling design of conjugate M1 formed by gradual hybridizations of a 15 nm monofunctionalized conjugate B and strands A/A1/B1. From the gel results, it was clear that the gel bands of conjugating products were generated with gradual, slow running speeds. In this system, the DNA/AuNPs conjugates of A1d-NP (10 nm multivalent AuNP) and Adx-NP (5 nm multivalent AuNP) were prepared, containing the same functional DNA sequences as inputs A1d and Ad, respectively. As shown in Figure 5b, in the presence of 10 nm A1d-NP, the ring A+A1 was opened to hybridize with it, thus forming conjugate M1-a carrying two gold nanoparticles of 15 and 10 nm. From the gel results of lane 3 in Figure 5c, it was easy to recognize a newly generated product band (purple arrow), indicating the dimer M1-a (also seen in Figure S5a, Lane 1). However, by only adding Adx-NP to conjugate M1, the gel results showed that almost no displacement product M1-b (consisting of two gold nanoparticles of 10 and 5 nm) was generated (Figure 5c Lane 4). This phenomenon was different from the PAGE gel results that Ad alone could directly result in the two rings separation of product C2. The possible reason is that the strong repulsion among gold nanoparticles prevented strand displacement occurring in the interlocked DNA/AuNPs clusters.

Figure 5. Programming monovalent DNA/AuNP conjugate M1. (a) Gradual assembly of conjugate M1, and results of formations on a 3% agarose gel. (b) Designs of strand displacement


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manipulations of programmed M1 structures. (c) Gel results of the assembling operations and TEM images. (d) Statistical analysis of the TEM images. Interestingly, when adding both Adx-NP and A1d-NP to trigger the M1 conjugates, a new gel band of AuNPs dimer M1-b was generated as shown in Figure 5c lane 5 (green arrow). For comparison, when both Adx (DNA strand only) and A1d-NP were present, the dimer M1-a in lane 6 disappeared when compared with the gel results in lane 5, indicating the complete separation of 10 and 5 nm AuNPs of M1-a. In addition, to confirm the target DNA/AuNPs clusters in the specific bands, all product bands of AuNPs bands were purified and counted from the TEM results. The TEM results confirmed the programmed nanoparticles configurations as shown in Figure 5c (more TEM images can be found in Figures S6). From the statistical analysis in Figure 5d, the production of dimer M1-a was 45.5% and dimer M1-b was 58% (M1-a from 110 and M1-b from 157 samples). Meanwhile, high yields of undesired monomers were also generated during production of M1-a and M1-b, at 37.3% and 26.8%, respectively. The possible reasons for the relative low production of targets may (1) complex nanoparticle configurations of the targets; (2) drying of the carbon plate; (3) the incomplete band separation; and (4) the manual collection operations. Although the yields of purified products were not very high, the statistical results still demonstrated generations of the designed structures. Conclusion In this study, DNA sequential logic gate was established using two-ring DNA, which can recognize two signals with different triggering sequences. In the logic operations, a “loop-open” mechanism was introduced to control the complete separation of the two-ring DNA. The sequential logic gate was implemented by PAGE gel and fluorescent detection. The “sequential


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effects” in treatments of DNA signals were recognized by comparing specific fluorescent outputs. In addition, the programmed nanoparticle arrangement guided by two-ring DNA was also constructed. We envision that, combined with other recent developed sensing methods, the resulting sequential logic gate will have further potential applications in related fields of gene engineering, diagnosis, and analysis. ASSOCIATED CONTENT Supporting Information. Materials, experimental methods, and additional experimental data. This material is available free of charge via the internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *To whom correspondence should be addressed. Email: [email protected], [email protected]; [email protected]; Present Addresses †If an author’s address is different than the one given in the affiliation line, this information may be included here. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT


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We thank Prof. H. Inaki Schlaberg for manuscript revision. The authors acknowledge financial support from National Natural Science Foundation of China (Grants 61272161, 61370099, 61425002, 61472333, 61320106005 and 61572046). REFERENCES 1.

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31. Zhang, D. Y.; Seelig, G. Dynamic DNA Nanotechnology Using Strand-Displacement Reactions. Nat. Chem. 2011, 3, 103-113. 32. Ciengshin, T.; Sha, R.; Seeman, N. C. Automatic Molecular Weaving Prototyped by Using Single-Stranded DNA. Angew. Chem. Int. Ed. 2011, 50, 4419-4422. 33. Ackermann, D.; Jester, S. S.; Famulok, M. Design Strategy for DNA Rotaxanes with a Mechanically Reinforced PX100 Axle. Angew. Chem. Int. Ed. 2012, 51, 6771-6775. 34. Elbaz, J.; Wang, Z. G.; Wang, F.; Willner, I. Programmed Dynamic Topologies in DNA Catenanes. Angew. Chem. Int. Ed. 2012, 51, 2349-2353. 35. Sahu, S.; LaBean, T. H.; Reif, H. J. A DNA Nanotransport Device Powered by Polymerase Φ29. Nano. Lett. 2008, 8, 3870-3878. 36. Schmidt, T. L.; Heckel, A. Construction of a Structurally Defined Double- Stranded DNA Catenane. Nano. Lett. 2011, 11, 1739-1742. 37. Lu, C. H.; Qi, X. J.; Cecconello, A.; Jester, S. S.; Famulok, M.; Willner, I. Switchable Reconfiguration of an Interlocked DNA Olympiadane Nanostructure. Angew. Chem. Int. Ed. 2014, 53, 7499–7503. 38. Cecconello, A.; Lu, C. H.; Elbaz, J.; Willner, I. Au Nanoparticle/DNA Rotaxane Hybrid Nanostructures Exhibiting Switchable Fluorescence Properties. Nano Lett., 2013, 13, 6275–6280. 39. Elbaz, J.; Cecconello, A.; Fan, Z. Y.; Govorov A.; Willner, I. Powering the Programmed Nanostructure and Function of Gold Nanoparticles with Catenated DNA Machines. Nat. Commun., 2013, 4, 2000-2007.


17


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Page 18 of 23

Ad Ad

1st FI

2nd FI A1d

TOC


18

Page 19 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

b B1

A1 1

B

A

A1

2

3

4

5

6

C1 Interlocked

B1

B1

A1

A

5-CATACTCTTTAG-3

12 bp hybridizing region

C2

C AB

Uninterlocked two-ring DNA

B

C

Figure 1


ABA1 ABB1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 23

a Ad loop-open Ad C1

S1 Intermediate State

Ad

A1d A1d

C2

S3 Seperated State 3

Ad

C two-ring DNA

S2 Seperated State 1

b

C

C2

C

C2

C

-

-

-

-

A1d A1d

-

-

Ad

Ad

Ad

Ad

3

4

5

6

7

8

two-ring DNA C 1

2

a

C2

9

+Ad-FAM +A1d

+Ad-FAM

+A1d

+A1d

AB

AA1-Ad-A1d AA1-Ad

a

BB1

a

+Ad-FAM +A1d

+A1d

+Ad-FAM +A1d

c

+A1d

C2

C

+Ad-FAM

C +Ad-FAM a C2

+A1d

+A1d+A1d

-

HEX

a-

1

3

2

-

A1d A1d

+A1d

+Ad-FAM +A1d

Ad

4

FAM

C2

C

+Ad-FAM

-

-

+Ad-FAM

Ad

+Ad-FAM

6

5

+A1d

Ad

+A1d

7

Ad +Ad-FAM

8

9

+Ad-FAM

+A1d

+A1d

a +Ad-FAM +A1d

+A1d

+Ad-FAM +Ad-FAM

+A1d

+Ad-FAM

+A1d

a

a

+A1d

+A1d

+Ad-FAM +A1d

+Ad-FAM

+A1d

+Ad-FAM +A1d

+A1d

+Ad-FAM +Ad-FAM +A1d+A1d

+Ad-FAM+Ad-FAM

+A1d

+A1d

+Ad-FAM

+A1d

Figure 2 +Ad-FAM

+Ad-FAM

+A1d

+Ad-FAM +Ad-FAM +A1d


+A1d

+A1d 10

Page 21 of 23

a

b Ad

C1

Ad HEX

S1

Ad

BHQ

1st FI

A1d

Ad

C2

A1d

2nd FI

2nd FI

A1d

S3 two-ring DNA

C

1st FI

S2

c

d

1st FI

Q value 3.5

2nd FI

3.07

3 2.5

2nd FI

Fluorescent intesity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A1d

1st FI

Pathway 1

2

Pathway 3

1.5

TAE buffer

1.69

0.39

0.5

0.01 pathway 1

Ad

Table 1

Ad

A1d 6

12

18

24

2.33

1 Pathway 2

Ad

2.37

30

36

42

48

54

(min)

Figure 3 ACS Paragon Plus Environment

input 1

pathway 3

pathway 2 input 2

Pathway 1

Ad

A1d

Pathway 2

Ad

---

Pathway 3

A1d

Ad

1st FI

2nd FI

-----


A1d

Ad

C P1

Fluorescent intesity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 23

P2

P1

P2 6

12

18

24

30

Figure 4


36

42

48

54

(min)

Page 23 of 23


15 nm 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

b

A1

B1

15 nm 5 nm

Adx-NP

5 nm

15 nm

5 nm

B1

A1d-NP A

B

B

1

B1

A1 B1

1

A1 1

A1d-NP

10 nm

5 nm

B1

M1-b

A1

A1d-NP

Adx-NP 15 nm

10 nm

5

4

3

Adx1

1 Adx-NP

M1-a

5 nm

5 nm

A1d-NP

B1

5 nm

A1d-NP

A1

10 nm

Adx-NP

5 nm 15 nm

Adx

15 nm

15 nm

Adx 1

5 nm

M1

15 nm

10 nm

5 nm

10 nm

5 nm

5 nm

15 nm

A1

15 nm

5 nm

5 nm

A

2

Adx-NP

15 nm

15 nm

15 nm

A1

B1

1

M1 5 nm

5 nm

Adx-NP

B1

A1

M1

15 nm

15 nm 15 nm

15 nm 15 nm

15 nm

15 nm

Adx-NP Adx A1d-NP A1d-NP A1d-NP Adx-NP Adx-NP

c

A1dNP-sh

M1 1 15 nm

15 nm

15 nm 15 nm

1515 nm nm

5 nm

2

M1 3

M1 4

M1 6

M1 5

5 nm

15 nm

10 nm

A1dNP-sh

5 nm

Adx-NP

7

8

15 nm

15 nm

15 nm 15 nm

nm

d

15 nm

15 nm 15 nm

5 nm

15 nm

60%

M1-a 45.5%

40%

5 nm

15 nm

37.3% 10 nm

5 nm

20%

10%

A1d-NP 5 nm

7.3%

M1-b monomer

Percentage of different polymer structures (%)

m

Percentage of different polymer structures (%)

B1

target

trimer

aggregated derivatives

M1-a M1-a

M1-b

50 nm

58%

60%

Adx-NP

M1-b

40%

26.8% 20%

12.7% 2.5% monomer

target

trimer

aggregated derivatives

50 nm

Figure 5


50 nm

DNA Sequential Logic Gate Using Two-Ring DNA - ACS Applied

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