Programmable Regulation of DNA Conjugation to Gold Nanoparticles

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Programmable Regulation of DNA Conjugation to Gold Nanoparticles via Strand Displacement Cheng Zhang, Ranfeng Wu, Yifan Li, Qiang Zhang, and Jing Yang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02620 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 6, 2017

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Programmable Regulation of DNA Conjugation to Gold Nanoparticles via Strand Displacement Cheng Zhang, a †＊ Ranfeng Wu,b † Yifan Li, b Qiang Zhang,c＊Jing Yangb＊ a

Institute of Software, School of Electronics Engineering and Computer Science, Peking

University. Key laboratory of High Confidence Software Technologies, Ministry of Education, Beijing 100871, China. Email: [email protected] b

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

Beijing 102206, China. Email: [email protected] c

College of Computer Science and Technology, Dalian University of Technology, Dalian,

116624, China. Email:[email protected] †

C.Z. and R.W. contributed equally.

KEYWORDS. DNA detection; DNA computing; DNA/AuNPs conjugation; Catalytic circling; Strand displacement

ABSTRACT. Methods for conjugating DNA to gold nanoparticles (AuNPs) have recently attracted considerable attention. The ability to control such conjugation in a programmable way is of great interest. Here, we have developed a logic-based method for manipulating the conjugation of thiolated DNA species to AuNPs via cascading DNA strand displacement. Using this method, several logic-based operation systems are established and up to three kinds of DNA signals are introduced at the same time. In addition, a more sensitive catalytic logic-based operation is also achieved based on an entropy-driven process. In the experiment, all of the DNA/AuNPs conjugation results are verified by agrose gel. This strategy promises a great

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potentiality for automatically conjugating DNA stands onto label-free gold nanoparticles and can be extended to constructing DNA/nanoparticle devices for applications in diagnostics, biosensing and molecular robotics.

Introduction DNA, by virtue of its abundant information content, ability to hybridize specifically, and the ease of its preparation, has emerged as a leading material for building various nanostructures1-10. Over the past two decades, researchers have tried their best to couple DNA to many materials, including gold nanoparticles (AuNPs)

11-16

, carbon nanotubes17-20, and proteins

21-23

, in which

DNA is used as both an information carrier and as a scaffolds for constructing functional nanodevices. In particular, the conjugation of DNA and AuNPs has attracted considerable attentions for the ease of operability and its programmability. By accurately regulating specific DNA strands conjugating with AuNPs, one can better control the arrangement and aggregation of nanoparticles in a programmable way24-26. Currently, many researchers have performed the conjugations to construct various AuNPs assembling nanostructures 27,28. Nonetheless, almost all such conjugations proceed straightforwardly: thiolated DNA can directly attach onto the surface of nanoparticles without switchable controls29-31. Accordingly, very few hierarchical control over the conjugation of DNA to AuNPs has been reported to date. In fact, a major challenge in the hierarchical DNA/AuNPs conjugation is specific control of the thiol-group activity. To this end, we recently developed a method that permits selective control the mono-conjugation of DNA to AuNPs by manipulating thiolated hairpin DNA32.


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On the other hand, DNA strand displacement is a self-driving reaction to achieve the lower energy states based on specific hybridizations

33,34

. Through catalytic cycling pathways to

recognize DNA molecules, the mechanism has been widely applied to constructing molecular sensing systems and nano-devices.33,35 Notably, such cascading displacements are adapted to analyze multiple DNA targets by introducing complex up and down stream recognizing sites. Meanwhile, this method also possesses several other advantages such as higher specificity, ease of control and enzyme-free catalytic circuits. Thus, the incorporation of the cascading strand displacing reaction into the label-free DNA/AuNPs conjugation systems may yield an efficient and programmable DNA/gold nanoparticle assembly. Actually, combined with DNA strand displacement, a wide range of systematically organize nanoparticles structures have emerged as an ideal tool for applications related to catalytic and sensing fields.

36-39

In particular,

fundamental studies focus on the DNA assisted nanoparticle plasmonic motif due to its versatile assembly structure. 40,41

In the present study, a logic-based DNA/AuNPs conjugating system is established, exploiting the combination of conjugations of DNA/AuNPs and the programming DNA strand displacement. In the experiment, all of the conjugation results are verified by agrose gel. In the reaction, the attachments of DNA onto AuNPs are achieved in a hierarchical and logic-based way and at the single-molecule level. In addition, the control of enzyme-free catalytic circuit was also constructed as a functional unit to analyze multiple DNA targets, in which the concentration requirement of DNA regulators becomes much lower than that of other logic-based operations. This strategy promises a great potentiality for automatically conjugating multiple DNA signals onto label-free gold nanoparticles for applications in diagnostics, biosensing and molecular robotics.


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Experimental Section Materials. The Gold nanoparticles (AuNPs) were purchased from Ted Pella, and BSPP salt (Bis (p-sulfonatophenyl) phenyl phosphine dipotassium salt dehydrate) was purchased from Strem Chemical. In addition, the Tris-Borate-EDTA buffer of pH8.0 (TBE) and Tris-Acetic-EDTA buffer of pH8.0 (TAE) were from Solarbio; unmodified DNA strands and thiolated DNA were purchased from Sangon China by HPLC purification. The agarose gel was from BIOWEST. In Polyacrylamide gel electrophoresis gel (PAGE), methylenebisacrylamide and acrylamide monomer

was purchased from TCI; Stains-All was purchased from SIGMA ALDRICH. DNA assembly. To assemble the designed DNA scaffolds, a reaction with volume of 80 µL 1×TAE buffer was used (containing 12.5mM MgCl2). Each DNA strand was hybridized at room temperature with a concentration of 3 µM for 2 hours. Then, the DNA products were detected by 10% non-denaturing PAGE gels for about 2-3 hours at an electric field of 5-10 V/cm under room temperature. Finally, all DNA molecules were labeled by StainAll and recorded by a Canon scanner. The gel experiment was performed by a mini gel device (Bio-Rad) in 1×TAE buffer (containing 12.5mM MgCl2). Phosphination of AuNPs. Before conjugating DNA and AuNPs, the particles should be protected by BSPP to increase the stability in solution. The BSPP solid powder was mixed with AuNPs in the ratio of 25ml to 12.5mg. Then, the reaction was shaken overnight at room temperature. After the shaking course, NaCl powder was slowly added into the solution with continuous stirring, with the color of the solution changing from red to purple. The color changed solution was centrifuged at 4000~7000 rpm for 15 minutes with the supernatant fluid


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being discarded. Finally, the protected AuNPs sample was diluted in a 300 µL 1×BSPP solution (for 10×BSPP solution: 1.24 mg BSPP dissolve in 1 mL deionized water). Monovalent conjugation of DNA and AuNPs. To perform the monovalent conjugation, in a typical conjugating experiment, 5 nm AuNPs were used with the concentration of 3 µM and each of the DNA concentrations being 1.5 µM. Notably, the conjugation is very sensitive to the ratio of thiolated DNA and AuNPs. With the increment of the ratio, multiple DNA strands can bind with AuNPs, which will lead to a slow running mobility of the conjugating products. The reaction was implemented in a 0.5×TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0) with a NaCl concentration of 50 mM. With a 4-6 hours incubation, the triggered thiolated DNA strands can conjugate with the AuNPs. The conjugations were detected by 3% agarose gel (running gel buffer 0.5×TBE, 70V, 1.5 hours), by which different DNA/AuNPs conjugates can be separated and discriminated with their individual gel running speeds. In addition, the product can also be purified by agarose gel using a glass fiber filter membrane supported by a dialysis membrane (MWCO 14000) and preserved at 4°C. The concentrations of gold nanoparticles were detected using optical absorbance at 520 nm. Hybridization of nanoparticles dimers. To perform the hybridization, mulitivalent conjugate γ was prepared to connect with the monovalent conjugate β. In a typical reaction, 4-8 µL of purified conjugate γ was mixed with monovalent 5 nm conjugates β. Then, the hybridizing reactions were incubated at room temperature for 4-6 hours. Finally, the results were detected by using 3% agarose gels for 1-2 hours (0.5×TBE running buffer). TEM Analysis. The purified conjugating product solution (5 µL) was deposited on carbon grids. Then, the excess solution was removed with filter paper. Finally, the grids were washed 1-3 times with Milli-Q water and air dried for 5-10 min.


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Results and Discussion The basic principle of the hierarchical control of the attachment, is illustrated by the example of a one-level “YES” gate. In this experiment, all logic-based operating results are verified by the generation of target product band detected by a gel image system. As shown in Figure 1, upon only introducing of thiolated hairpin B directly to 5 nm AuNPs, no conjugation occurs. This is because of spatial hindrance from the concave end of the double-stranded DNA, which restricts thiol groups to connect onto the surface of the AuNPs. The conjugating result was confirmed by the generation of target gel band in Lane 1 (Figure 1b), where no band was generated for the conjugating product when only thoilated hairpin DNA and AuNPs were mixed (also seen in Figure S3). While in the presence of signal SAS (a thiolated DNA species), SAS can directly conjugate with AuNPs via the thiol-groups. Meanwhile, SAS can also hybridize with hairpin B, thereby bringing the thiol groups of hairpin B into close proximity to the AuNP surface. In this case, the attachment of hairpin B onto AuNPs can occur and conjugate α1 is generated consequently. In Figure 1b Lane 2, a clear band for product α1 can be seen from the gel results. Notably, when the concentration of signal SAS increased from 0.75 µM to 1.5 µM, the product increased accordingly (Lane 3, Figure 1b). a B

B

B

B SAS

1 5-ACAAGCGACGA AGCAGGG-3

“Inactive” thiol-group

conjugate α1 B

b

SAS

1

1

B B SAS SAS 0.75 µM 1.5 µM 2

3

YES

conjugate α1


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Figure 1. (a) Illustration of “inactive” thiolated hairpin DNA and the principle of one-level “YES” gate. (b) Conjugation verified by 3% agarose gel electrophoresis. In a typical experiment, the concentration of 5 nm label-free AuNPs was 3 µM and that of each DNA species was 1.5 µM.

To further expand on our ability to manipulate conjugation, a two-level cascading “YES” gate was constructed (Figure 2). In this system, the reactivity of the thiol group on strand SAS is initially hindered by sequestration of the thiol group at the SAS/SA1 double-stranded concave end. Only in the presence of strand SA2 (having longer complementary regions with SA1 than SAS), can signal SAS be released, initiating the conjugation of DNA to AuNPs. As confirmed by Lane 3 in Figure 2b, without the addition of strand SA2, no band was generated for the conjugation product. In the experiment, when strand SA2 was introduced, a band was generated for the conjugation product (conjugate α1; Figure 2b, Lane 6), as anticipated. To further compare the effects of thiol group conjugating with AuNPs, an additional control experiment was also implemented, in which a non-thiolated hairpin B1 was used. As expected, in the presence of SAS and hairpin B1, SAS brings B1 into proximity with AuNPs; however, because B1 lacked a thiol group it was not able to conjugate to AuNPs. Interestingly, the small modification difference between hairpin B and B1 produced a great change in the rate of gel migration between the resulting conjugation product bands (Lanes 4 and 5, Figure 2).


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a B

B B

SAS

B

3’ 5’

2 5’

SA2

3’

3’

conjugate

α1

3’

SA1

1

b B

B

SA2

YES

1

SAS SA1

SAS SA1

2

3

SAS B1

SAS B

SA2 B SAS SA1

4

5

6

SAS

YES conjugate α1

Figure 2. (a) Illustration of a two-level cascading “YES” gate. (b) 3% agarose gel electrophoresis results. SAS: Thiolated linear DNA. B: Thiolated hairpin DNA. B1: Non-thiolated hairpin DNA. In a typical experiment, 5 nm label-free AuNPs are used with a concentration of 3 µM and each of the DNA concentrations is 1.5 µM. DNA strands in Lane 1 to 6: B, SAS/SA1, B+SAS/SA1, SAS+B1, SAS+B, SAS/SA1+B+SA2, respectively. Furthermore, a two-input “OR” gate was also implemented to test the feasibility and parallelism of this strategy, in which the conjugation can be triggered in the present of either input C1 or C2. In this “OR” gate, thiolated strand ‘Cs’ is initially protected by its hybridization with strand Cs1, which contains two specific recognition toehold sites on both ends, to recognize DNA input signals. Therefore, in the presence of either input C1 or C2, the thoilated DNA Cs can be released, thus resulting in the binding assistance attachment and the generation of


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conjugate α2. These logic-based conjugating manipulations were verified by agarose gel electrophoresis (Figure 3b) and DNA assembly results can be found in Figure S3; the addition of either C1 or C2 generated a band for newly formed product. a 2

E

C1

E

E

3’

Cs 3’ 3’

5’

1

5’

5’

C2

Cs1 conjugate α2

b

C2

C2

3

C1 4

C1 1

2

E

3’

C1

5’

OR C2

conjugate α2

Figure 3. (a) Illustration of a two-input “OR” gate. (b) Results from 3% agarose gel electrophoresis. CS: Thiolated linear DNA. E: Thiolated hairpin DNA. In a typical experiment, 5 nm label-free AuNPs are used with a concentration of 3 µM and each of the DNA concentrations is 1.5 µM. DNA strands in Lane 2 to 4: C1+E+Cs/Cs1, C2+E+Cs/Cs1, C1+C2+E+Cs/Cs1, respectively.

To characterize the performance of our novel strategy in detail, an enzyme-free DNA catalytic cycle was also introduced to control the conjugation reactions and to construct an “AND” gate. Similar to the mechanism described above, the thiolated DNA hairpin HF is initially “protected”


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through a hairpin DNA assembly structure (HDA), the spatial hindrance of which inhibits direct binding between the thiol groups and AuNPs. To trigger the conjugation, both input signals ‘DE’ and ‘BCD’ need to be introduced at the same time, performing an “AND” operation. During the first step of the catalytic process, input DE binds to the single-stranded toehold domain of HDA, displacing strand CD and forming an intermediate product (I-HDA) with an exposed 4 bp ‘site C’ in the middle region (Figure 4a). However, this intermediate product is not stable enough in the presence of the input signal BCD. Therefore, the newly exposed site C can facilitate the hybridization of input BCD and A-E-1, resulting in rapid rearrangements that disassemble IHDA. Consequently, DNA molecules of DE and (HF+AB) are released from the initial hybridized structures. It should be noted that, only in this case, the activity of the thiol-group of AB) is restored, thus triggering the conjugation of (HF+AB) to AuNPs. Moreover, the released DNA strand DE can re-participate in the next strand displacement cycles, enabling loss-free regeneration. It is noteworthy that DE acts both as an input signal to initiate the conjugation of DNA onto AuNPs, and as a regenerated catalyst in the strand displacement cycles. As a consequence, only a small amount of the catalyst DE is required to produce a large number of active thiol group-bearing AB molecules, greatly increasing the rate of conjugation of DNA to AuNPs. In our experiments, strands DE and BCD are used as input signals to implement an “AND” gate, as depicted in Figure 4a. It can be clearly seen that adding either input separately (DE or BCD), does not trigger the formation of any obvious conjugate δ (Figure 4b, Lanes 3 and 4). However, when both inputs are added at the same time, the conjugation reaction is triggered, resulting in a new gel band containing conjugate δ (Figure 4b, Lane 5; and Figure S6). Subsequently, to further demonstrate the catalytic effects of this “AND” gate, two control


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experiments were carried out by varying the concentrations of DNA signals. In control experiment 1, we sought to examine the role of the fuel strand BCD in the enzyme-free circuit. In our design, we used a constant concentration of 6 µM for the DNA strands HDA and DE. Meanwhile, the concentrations of BCD were set to 0.06 µM, 0.3 µM, 1.2 µM, or 6 µM. Note, however, that the rather lower concentrations of BCD proved unable to induce a significant product band for conjugate δ (Figure 4c, Lanes 2 to 4). As indicated by the red arrow, many free AuNPs remained unconjugated. A distinct product band containing conjugate δ was only generated at 6 µM BCD, which was the highest concentration tested (Figure 4c, Lane 5). In control experiment 2, which sought to quantitatively characterize the performance of catalyst DE. Here, the concentrations of DE were set to 0.06 µM, 0.3 µM, 1.2 µM, or 6 µM. Meanwhile, a constant concentration (6 µM) was used for the DNA species HDA and BCD, to guarantee the availability of sufficient fuel strands for the circuits. Interestingly, as the DE concentrations increased, a distinct gel band for conjugate δ became visible (Figure 4d, Lane 2), starting at 0.3 µM. In addition, to verify the reliability of the catalytic cycling detection, the control experiment 3 that was lacking one input signal was also implemented as shown in Figure S7. Even treated by high concentration up to 60 µM, no target conjugation was generated with only single input. Of particular interest, when one compares the conjugation results obtained using non-catalyzing BCD and the catalyst DE (at the same concentrations), the number of unreactive free AuNPs is much lower when using the catalyst DE (indicated by the blue arrow) than it is when using the fuel strand BCD (red arrow). We infer that, when using an enzyme-free catalytic strategy, our process results in a higher strand displacement rate and more products from conjugation reactions between DNA and AuNPs. Collectively, these experimental results clearly demonstrate


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that, using this conjugation control system, only a small quantity of catalyst suffices to trigger the

entire

program

reactions.

a

HF

C 4bp exposed site DE

of

BCD

DE HF 1 AB

HF

CD Catalytic cycle

A-E-1 Hairpin DNA Assembly (HDA)

A-E-1

DE HF

HF

3’

AB

regenerated 3’

2

BCD

5’

AB

3’

A-E-1

3’

5’

conjugate δ b c

BCD

AND

DE

(HDA)

BCD + (HDA)

DE + (HDA)

BCD

d

1

2

3

4

0.06 µ

0.3 µ

1.2 µ

4

6µ

BCD + DE + (HDA) 1

2

3

DE

0.06 µ

0.3 µ

5

1.2 µ

6µ

3

4

5 1 conjugate

2

δ

Figure 4. (a) Schematic overview of the logic-based manipulations in the DNA catalytic cycle. DNA strands HF and AB are both modified with thiol groups at their 3ʹ ends. (b) Conjugation was verified by 3% agarose gel electrophoresis. AB: Thiolated linear DNA. HF: Thiolated hairpin DNA. In a typical experiment, 5 nm label-free AuNPs are used. DNA strands in Lane 2 to 4: HAD structure, BCD+HAD structure, DE+HAD structure, BCD+DE+HAD structure,


12

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respectively. Panels (c) and (d) display the gel results for when the concentrations of BCD and DE were increased. These concentrations are arranged: 0.06 µM, 0.3 µM, 1.2 µM, and 6 µM. In this experiment, 5 nm label-free AuNPs were used (3 µM) and one of the other DNA concentrations was 6 µM.

Conclusion In summary, we have developed a method to achieve programmable mono-conjugation to AuNPs controlled by multiple DNA signals. By taking advantage of cascading DNA strand displacement and triggered binding-assisted attachment, we were able to perform logic-gated functions by using regulator DNA and confirm the cascading features of the system. In addition, to better understand cascading circuits, a more sophisticated catalytic conjugation system has been established using an enzyme-free cyclic process, in which the advantages as high sensitivity and recyclable circuits are well demonstrated. Considering its programmability, catalytic activity, and specificity, this strategy holds great promise for automatically controlling DNAAuNP nanostructures, and it could potentially be extended to the construction of other nanoscale systems with applications in diagnostics and bio-sensing

ASSOCIATED CONTENT Supporting Information. Description of other experimental procedures and additional figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding authors


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* To whom correspondence should be addressed. Email: [email protected]; [email protected]; [email protected] Author Contributions The study was designed by Cheng Zhang, and was carried out by Ranfeng Wu and Yifan Li. The manuscript was written by Cheng Zhang, Jing Yang and Qiang Zhang. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT The authors acknowledge financial support from National Natural Science Foundation of China (Grants 61672044, 61370099, 61425002, 61472333, and 61320106005). the Fundamental Research Funds for the Central University, 2016MS46 REFERENCES 1.

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with

nanometer

resolution

via

backbone-modified

phosphorothioate DNA and bifunctional linkers. J. Am. Chem. Soc. 2010, 132,8906-8908 23. Voigt, N. V.; Tørring, T.; Rotaru, A.; Jacobsen,

M. F. Single-molecule chemical

reactions on DNA origami. Nat Nanotech. 2010, 5, 200-203 24. Pal, S.; Sharma, J.;Yan, H.; Liu, Y. Stable silver nanoparticle – DNA conjugates fordirected self-assembly of core-satellite silver – gold nanoclusters. Chem. Commun. 2009, 45, 6059-6061 25. Thomas, G. W. E.; Kai, L. L.; Danny, B.; Christopher, J. S.; Hanadi, F. S. Transfer of molecular recognition information from DNA nanostructures to gold nanoparticles. Nature Chem. 2016, 8, 162-170


17

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26. Mathew, M. M.; Mudalige, T. K.; Dmytro, N.; William, B. S.; Oleg, G. Switching binary states of nanoparticle superlattices and dimer clusters by DNA strands. Nature Nanotech. 2010, 5, 116-120 27. Claridge, S. A.; Goh, S. L.; Frachet, J. M. J.; Williams, S. C.; Micheel, C. M.; Alivisatos, A. P. Directed assembly of discrete gold nanoparticle groupings using branched DNA scaffolds. Chem Mater. 2005, 17, 1628-1635 28. Song, S.; Liang, Z.; Zhang, J.; Wang, L.; Li, G.; Fan, C. Gold-nanoparticle-based multicolor nanobeacons for sequence-specific DNA analysis. Angew. Chem. Int. Ed. 2009, 48, 8670-8674 29. Schreiber, R.; Do, J.; Roller, E.; Zhang, T.; Schuller, V. J.; Nickels, P. C.; Feldmann, J.; Liedl, T. Hierarchical assembly of metal nanoparticles, quantum dots and organic dyes using DNA origami scaffolds. Nature Nanotech. 2014, 9, 74-78 30. Kuhler, P.; Roller, E.; Schreiber, R.; Liedl, T.; Lohmuller, T.; Feldmann, J.Plasmonic DNA-Origami Nanoantennas for Surface-Enhanced Raman Spectroscopy. Nano Lett. 2014, 14, 2914−2919 31. Pilo-Pais, M.; Goldberg, S.; Samano, E.; LaBean, T. H.; Finkelstein, G. Connecting the Nanodots: Programmable Nanofabrication of Fused Metal Shapes on DNA Templates. Nano Lett. 2011, 11, 3489–3492 32. Zhang, C.; Ma, J. J.; Yang, J.; Liu, S.; Xu, J. Binding assistance triggering attachments of hairpin DNA onto gold nanoparticles. Analytical chem. 2013, 85, 11973-11978


18

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19

Langmuir

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 25

41. Jiang, Q.; Wang, Z. G.; Ding, B. Q. Programmed Colorimetric Logic Devices Based on DNA–Gold Nanoparticle Interactions. Small 2013, 9, 1016-1020

TOC GRAPHICS

B

B

B SAS


SAS

conjugate α1

YES


20

Page 21 of 25

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

Langmuir

a

B

B

B

B SAS 1

5-ACAAGCGACGA AGCAGGG-3


conjugate α1 B

b

SAS

1

1

B B SAS SAS 0.75 µM 1.5 µM 2

3

YES

conjugate α1 ACS Paragon Plus Environment

Langmuir

a

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

B

Page 22 of 25

B B

SAS

B

3’ 5’

2 SA2

5’

3’

3’

conjugate

α1

3’

SA1

1

b

B

B

SA2

YES

1

SAS SA1

SAS SA1

2

3

SAS B1

SAS B

SA2 B SAS SA1

4

5

6

SAS

YES conjugate α1 ACS Paragon Plus Environment

Page 23 of 25

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

Langmuir

a

2

E

C1

E

E

3’

Cs 3’ 3’ 5’

5’

1

5’

C2

Cs1 conjugate α2

b

C2

C2

3

C1 4

C1 1

2

E

3’

C1

5’

OR

C2

conjugate α2


Langmuir

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

HF

Page 24 of 25

C 4bp exposed site DE

BCD

DE HF 1 AB

CD

Catalytic cycle

A-E-1 Hairpin DNA Assembly (HDA)

AB

A-E-1

DE

HF

HF

regenerated 3’

2

3’

HF

5’

BCD

AB

3’

A-E-1

3’

5’

conjugate δ b c

BCD

AND

DE

(HDA)

BCD + (HDA)

DE + (HDA)

BCD

2

3

4

0.3 μ

1.2 μ

6μ

4

5

BCD + DE + (HDA)

d

1

0.06 μ

1

2

3

DE

0.06 μ

0.3 μ

1.2 μ

6μ

3

4

5 1 conjugate ACS Paragon Plus Environment

δ

2

Page 25 of 25

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

Langmuir

B

B

B SAS


SAS

conjugate α1

YES ACS Paragon Plus Environment

Programmable Regulation of DNA Conjugation to Gold Nanoparticles

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