PolyA-Mediated DNA Assembly on Gold Nanoparticles for

Subscriber access provided by UNIV OF CONNECTICUT

Article

PolyA-mediated DNA assembly on gold nanoparticles for thermodynamically favorable and rapid hybridization analysis Dan Zhu, Ping Song, Juwen Shen, Shao Su, Jie Chao, Ali Aldalbahi, Ziang Zhou, Shiping Song, Chunhai Fan, Xiaolei Zuo, Yang Tian, Lianhui Wang, and Hao Pei Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00891 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 9, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 19

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

Analytical Chemistry

PolyA-mediated DNA assembly on gold nanoparticles for thermodynamically favorable and rapid hybridization analysis Dan Zhu1,2†, Ping Song2†, Juwen Shen3, Shao Su1, Jie Chao1, Ali Aldalbahi4, Ziang Zhou5, Shiping Song2, Chunhai Fan2*, Xiaolei Zuo2, Yang Tian3, Lianhui Wang1, Hao Pei3* 1

Institute of Advanced Materials, Nanjing University of Posts and Telecommunications, Nanjing

210023, P. R. China 2

Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility,

Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, P. R. China 3

School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, P.

R. China 4

Chemistry Department, King Saud University, Riyadh 11451, Saudi Arabia

5

Johns Hopkins University, Baltimore, MD 21211 USA

To whom correspondence should be addressed. Tel: (+86) 021-54345484, E-mail: [email protected]. Tel: (+86) 021-39194127, E-mail: [email protected].

ACS Paragon Plus Environment


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

Abstract Understanding the behavior of biomolecules on nano-interface is critical in bioanalysis, which is great challenge due to the instability and the difficulty to control the orientation and loading density of biomolecules. Here, we investigated the thermodynamics and kinetics of DNA hybridization on gold nanoparticle, with the aim to improve the efficiency and speed of DNA analysis. We achieved precise and quantitative surface control by applying a recently developed poly adenines (polyA)-based assembly strategy on gold nanoparticles (DNA-AuNPs). PolyA served as an effective anchoring block based on the preferential binding with the AuNP surface and the appended recognition block adopted an upright conformation that favors DNA hybridization. The lateral spacing and surface density of DNA on AuNPs can be systematically modulated by adjusting the length of polyA block. We found the stability of duplex on AuNP was enhanced with the increasing length of polyA block. When the length of polyA block reached to 40 bases, the thermodynamic properties were more similar to that of duplex in solution. Fast hybridization rate was observed on the di-block DNA-AuNPs and was increased along with the length of polyA block. We consider the high stability and excellent hybridization performance come from the minimization of the DNA-DNA and DNA-AuNP interactions with the use of polyA block. This study provides better understanding of the behavior of biomolecules on the nano-interface and opens new opportunities to construct high-efficiency and high-speed biosensors for DNA analysis.

Introduction


Page 2 of 19

Page 3 of 19

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


DNA-nanoparticle conjugates, that combine the physical properties of inorganic nanomaterials and the biochemical specificity of DNA to realize unique functionalities, have attracted intensive interests in biosensing1-4, bioimaging5-9, therapy10-12 and nanophotonics13-15. Most recently, DNA gold nanoparticle (DNA-AuNPs) conjugates have been successfully employed to assemble various nanoparticle superlattices16,17. The biological investigations revealed that the superstructures assembled with DNA-AuNPs can improve their accumulation in tumors and accelerate the elimination from the body18. The tremendous growth in applications

requires

an

in-depth

and

quantitative

understanding

of

the

thermodynamics and kinetics of DNA hybridization on nanoparticles. The DNA gold nanoparticle-based biological assays have been widely used as sensitive tracers for the optical, electronic, acoustic and mechanical transduction of specific bio-molecular binding19-24/.]-\. Previous reports have demonstrated that the DNA gold nanoparticle based biological assays offer more significantly improved sensitivity over conventional analytical tools, for instance, the DNA gold nanoparticle bio-barcode PSA assay25 (~300 times more sensitive compared to commercial immunoassay), DNA assay with PCR-like sensitivity26,27. Although DNA hybridization has been extensively investigated in bulk solution and self-assembled monolayer on macroscopic solid surface, the increased complexity on nanoparticles presents new challenges to the fundamental understanding of DNA hybridization on AuNPs and the applications of DNA-AuNPs. Model systems using thiol-DNA AuNPs conjugates have shown that DNA conjugated on AuNPs has 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

higher binding constant than that of DNA in solution28. Some other investigations contradictorily demonstrated that DNA duplex on AuNPs became progressively less stable along with the hybridization process29,30. However, reproducibly and quantitatively anchoring of DNA on AuNPs still remains challenging, which impacts the DNA hybridization properties on AuNPs significantly. The employment of thiol-DNA and the following backfilling step is unlikely to control the dispersion of DNA on surface31. Imaging studies have revealed the heterogeneous surface packing density on thiol-DNA monolayer on gold surface32. We recently demonstrated the spatially controlled functionalization of AuNPs with designed di-block oligonucleotides, which would provide a good opportunity to investigate DNA recognition behavior on AuNPs with precise and quantitative control of DNA density and configuration33. Our di-block DNA-AuNPs were constructed based on the stronger adosorption of gold surface with adenine (A) bases than that with other three DNA bases. Consecutive adenines (polyA) serve as an effective anchoring block for preferential binding with the AuNP surface, and the appended recognition block adopts an upright conformation that favors the DNA hybridization33-35. The lateral spacing and surface density of DNA on AuNPs can thus be systematically modulated by adjusting the length of the polyA block. Here, by programmably tuning the length of the anchoring block (the number of adenines), we constructed di-block DNA-AuNP conjugates with controllable loading density and configuration. The thermodynamics and kinetics of DNA hybridization on AuNPs were programmably tailored (Figure 1). Our newly developed system provided deep


Page 4 of 19

Page 5 of 19

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


insight for understanding the behaviours of biomolecules on nano-interface.

Figure 1. Di-block DNA with consecutive adenines (anchoring block) and recognition probes (recognition block) can be immobilized on the AuNPs. The lateral spacing and surface density of DNA on AuNPs can be systematically modulated by tuning the length of anchoring block. Based on that, the thermodynamics and kinetics of DNA hybridization on AuNPs can be programmably tailored. EXPERIMENTAL SECTION: Chemicals and Materials. All oligonucleotides were synthesized and purified by TaKaRa Inc. (Dalian, China) and DNA sequences and their labeling are shown in Table S1. AuNPs (13 nm) were synthesized following the standard citrate reduction procedures. Briefly, tri-sodium citrate of 1% solution was added to a boiling, rapidly stirred solution of HAuCl4 (HAuCl4•4H2O, 99.9%, China National Pharmaceutical Group Corporation). After being kept boiling and stirred for 20 min, the solution was cooled to room temperature. The synthesized AuNPs were stored at 4 °C.



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

Preparation

of

Di-block

DNA

Modified

Page 6 of 19

Gold

Nanoparticles

or

Thiol-DNA-Modified Gold Nanoparticles. The prepared AuNPs were first incubated with di-block DNA or thiol-DNA (SH-DNA) in a 1:200 ratio for 16 h, 1M of sodium phosphate buffer (1 M of NaCl, 100 mM of Na2HPO4 and NaH2PO4, pH 7.4) was added to the mixture (a total of five salting steps with at least 30 min intervals between steps) to reach a final concentration of 0.1 M. The salting process was allowed by incubation for another 40 h at room temperature. Then, the mixture was washed 3 times with 0.1 M sodium phosphate buffer (PBS, 0.1 M of NaCl, 10 mM of Na2HPO4 and NaH2PO4, pH 7.4) through centrifugation (for 13 nm AuNPs, 13800 g, 20 min) to remove excess DNA and the nanoparticles were resuspended in 0.1M PBS (pH 7.4). The DNA hybridization detection indicated the specificity of DNA hybridization (Fig S2).

Quantification of Di-block-Oligonucleotide loaded on AuNPs. At first, di-block DNA (polyA05-P1-FAM, pol-yA10-P1-FAM, polyA15-P1-FAM, polyA20-P1-FAM, pol-yA30-P1-FAM, polyA40-P1-FAM)-AuNP were prepared following the above procedure. The concentration of AuNPs and the FAM-labeled diblock DNA in each sample were measured to quantify the number of DNA attached to each AuNP. The concentration of AuNPs was determined by UV-visible spectroscopy measurements and the absorbance values were related to the concentration of nanoparticles via Beer’s law. To determine the concentration of FAM-labeled DNA in each aliquot, the


Page 7 of 19

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


DNA was chemically displaced from the AuNPs’ surface using Mercaptoethanol (ME). The displacement was achieved by adding ME (final concentration 20 mM in 0.3 M NaCl 10 mM phosphate buffer solution (pH 7.4)) into the FAM-labeled DNA-AuNPs solution, which was then incubated for 18 h with shaking at room temperature. Released DNA probes were then separated via centrifugation and the fluorescence was monitored by a fluorescence spectrometer (F-4500, Hitachi, Tokyo, Japan). The fluorescence was converted to molar concentrations of probes by comparing to a standard linear calibration curve which was prepared using known concentrations of oligonucleotide with identical buffer pH, ionic strength and ME concentration. The number of oligonuleotides per AuNP was calculated by dividing the concentration of fluorescent oligonucleotides by the concentration of AuNP. All experiments were repeated three times using fresh samples to obtain error bars.

Melting Experiments. At first, di-block DNA (polyA05-P1, polyA10-P1, polyA15-P1,

polyA20-P1,

polyA30-P1,

polyA40-P1)-AuNP

and

thio-DNA

(SH-P1)-AuNP were prepared following the above procedure. Thermodynamic properties were measured by the fluorescence detection developed by Lytton-Jean and Mirkin28. Melting curves of dsDNA-AuNP from 2 nM to 20 nM were detected on a Fluorescence

spectrometer

equipped

with

a

temperature

controller.

The

di-block/thiol-DNA functioned 13 nm AuNPs were allowed to pre-hybridize with equal molar T1-FAM to form stable dsDNA-AuNP complex in 0.1 M PBS (10 mM of Na2HPO4 and NaH2PO4 containing 100 mM NaCl), pH 8.0 for 24 h. FAM was excited at 494 nm, and melting curves were recorded by monitoring emission



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 8 of 19

fluorescence at 520 nm in solution from room temperature to 70 °C at a linear heating rate of 20 °C/h.

Kinetic Analysis. For the hybridization rate measurement, equal amounts of di-block DNA

(polyA05-P1,

polyA10-P1,

polyA15-P1,

polyA20-P1,

polyA30-P1,

polyA40-P1)-AuNP / thiol-DNA (SH-P1)-AuNP and complementary FAM labeled T1 (T1-FAM) at 5 nM was mixed rapidly in 0.1M PBS (pH 7.4) at a temperature well below Tm and the fluorescence spectra were collected with time. The dissociation rates were determined by the label dilution technique developed by Morrison and Stols36. In brief, 5 nM AuNP-DNA (di-block or thiol-DNA) was allowed to pre-hybridize with equal concentration of complementary T1-FAM to form dsDNA-AuNPs. After equilibrium was reached, excess unlabeled T1 (500 nM) was added at low temperature. Time courses of dissociation were recorded by monitoring the fluorescence after the mixture was suddenly raised to a temperature to Tm.

Analysis of Thermodynamic and Kinetic Data. The thermodynamics analyses based on Tm values were determined by taking the maximum of the first derivation of a melting transition measured by fluorescence spectroscopy. The melting curve of each system is well fitted by the growth curve of Boltzmann function:

y = A2 + (A1 − A2)/(1 + exp ቀ

୶ି௫బ ௗ௫

ቁ)

1

By measuring the concentration dependence melting temperature, melting data such as the enthalpy and entropy of hybridization of each system can be calculated through equation 2, where CT is the total concentration of nanoparticles. Values of △G were


Page 9 of 19

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


calculated from equation 3.

1/ܶ௠ = ܴ/∆‫ ்ܥ݈݊ ∗ ܪ‬+ (∆ܵ − ܴ݈݊4)/∆‫ܪ‬

2

∆‫ ܪ∆ = ܩ‬− T∆ܵ

3

∆‫ = ܩ‬−RTln‫ܭ‬௘௤

4

The kinetic analysis was conducted to determine the initial reaction velocity of each system which was calculated from the fluorescence versus time data. Apparent activation energies of hybridization (Eah) and dissociation (Ead) are determined from the Arrhenius equation by a liner fit of ln k versus 1/RT in a suitable temperature range.

ln݇ = ln‫ ܣ‬− ‫ܧ‬௔ /ܴܶ

Results and discussion Based on the highly designable nature of our di-block DNA, we locally tuned the length of polyA block in di-block DNA to regulate the assembly of DNA on AuNPs. The surface density of DNA on AuNPs was quantified through the well-established method37. We found that the surface density on AuNPs (13 nm in diameter) gradually decreased along with the length increasing of polyA block (Figure S1 in Supporting Information), indicating the gradual increase of the distance between DNAs. Consistent with our previous studies33,38, the surface density of adenines on AuNPs was nearly independent of the length of polyA, indicating adenines can fully cover the binding sites on AuNPs.



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 10 of 19

Figure 2. (A) Melting curves of 5 nM probe to 5 nM fluorophore on AuNPs and in solution. For Au-thiol-DNA, Tm = 47.1 °C. For AuNP-di-block-DNA, Tm (AuNP-poly A10) = 47.2 °C, Tm (AuNP-poly A20) = 48.5 °C, Tm (AuNP-poly A40) = 49.8 ℃. For duplex in solution, Tm = 52.2 °C. (B) The melting temperature changes along with the regulation of DNA assembly. (C and D) Representative plots for the determination of DNA thermodynamics of different systems. All experiments were performed in 0.1 M PBS, where Tm was taken from fluorescence measurements.

Table 1 DNA hybridization thermodynamics in solution and on AuNPs System

△H （kcal/mol） △S（cal/mol ·K） △G（kcal/mol） △Gdiff（kcal/mol） Keq(M-1cm-1) at 298 K

Au-polyA5

-131.3

-370.1

-20.94

11.02

2.3 × 1015

Au-polyA10

-140.2

-398.4

-21.48

10.48

5.6 × 1015

Au-polyA15

-163.9

-470.9

-23.49

8.47

1.7 × 1017

Au-polyA20

-172.3

-496.9

-24.14

7.82

5.1 × 1017

Au-polyA30

-176.4

-508.8

-24.70

7.26

1.3 × 1018

Au-polyA40

-205.9

-598.5

-27.46

4.50

1.4 × 1020

Au-SH DNA

-147.4

-420.2

-22.09

9.87

1.6 × 1016

In solution

-246.3

-719.0

-31.96

/

2.7 × 1023


Page 11 of 19

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


By tuning the DNA surface density on AuNPs, we programmably regulated the thermodynamics of DNA hybridization on AuNPs. We examined the melting curve of DNA duplex in solution and on AuNPs. We observed that DNA duplex with thiol anchoring exhibited the lowest Tm value (47.1 °C) than the corresponding DNA duplex with polyA (Figure 2A), which suggested the relatively poor stability of DNA duplex with thiol anchoring. Surprisingly, we obtained gradually increased Tm values (from 47.2 °C to 49.8 °C) when we regulated the surface density of DNA by tuning the length of polyA, indicating the improved stability of DNA duplex on AuNPs. Along with the increase of polyA length, the Tm value approached to that of DNA duplex in solution (Figure 2A). The melting temperature increased along with the increase of polyA length, which indicated the more favorable base stacking of DNA duplex on AuNPs (Figure 2B). The broader melting range and the lower Tm value may originate from partially hybridized probes on surface, which can be effectively improved by increasing the polyA length. We then deduced the complete thermodynamic profiles through the analysis of melting curves (Figure 2C and D). As show in Table 1, the free energy of DNA duplex on AuNPs immobilized via Au-S interaction (-22.09 kcal/mol) is bigger than that of DNA duplex in solution (-31.96 kcal/mol), which translated into a free energy cost of 9.87 kcal/mol, indicating DNA duplex is less stable on the surface of AuNPs. Interestingly, we can improve the global stability of DNA duplex on AuNPs by regulating the polyA length. With the increase of polyA length from 5 to 40 oligonucleotides, the free energy cost decreased from 11.02 kcal/mol to 4.5 kcal/mol, which indicated the increase of the stability of DNA



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

duplex. The obvious improvement possibly originated from the resulting increase of the inter-distance between the DNA that eliminated the electrostatic repulsion between neighboring hybridized DNA strands (a primary reason for the destabilization of DNA duplex)39. Further analysis indicated that both favorable enthalpy and entropy changes contributed to the improvement. As a result, the apparent binding constant was enhanced for over 4 orders of magnitude with the increase of polyA length (Table 1).

Figure 3. (A) Real time monitoring the DNA hybridization on AuNPs for AuNP-SH-DNA and AuNP-polyA40, respectively. (B) The hybridization kinetics were programmable regulated by tuning the length of polyA block. (C) The rate constant of hybridization increased along with the increase of polyA length.

Table 2 The apparent activation energies of DNA hybridization thermodynamics in solution and on AuNPs System

Eah (kcal mol-1K-1)

AuNP-polyA5

24.40

AuNP-polyA10

20.76


Page 12 of 19

Page 13 of 19

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


AuNP-polyA15 AuNP-polyA20

19.19 16.56

AuNP-polyA40

14.15 14.48

AuNP-SH DNA

23.43

AuNP-polyA30

We next characterized the hybridization reaction kinetics systematically. By employing FAM-labeled target DNA and the super fluorescent quenching ability of AuNPs, we monitored the fluorescence change upon target hybridization on AuNPs to obtain the kinetic data. We then fitted the data to elucidate the changes in rate constant. We obtained a relatively low rate constant for the AuNPs modified with thiol-DNA (~0.003 s-1, Figure 3A). The nonspecific adsorption of thiolated single stranded DNA (ssDNA) to the AuNPs may slow down the hybridization rate. Additional steps such as backfilling with MCH would minimize the nonspecific adsorption and improve the hybridization kinetics. In a distinct contrast, our di-block DNA modified AuNPs significantly accelerated the hybridization kinetics (up to 7-fold increase) and the rate constants increased monotonically with the increase of polyA length (Figure 3B and 3C), indicating our ability to programmably regulate the hybridization rate. Our previous results proved that the recognition block of our di-block DNA on AuNPs adopted the upright conformation and extended towards the solution phase40-42, which minimized the barriers (e.g., molecular crowding and electrostatic repulsion) for hybridization. We determined the rate constants over a range of temperatures, through which we obtained the apparent activation energies (Eah in Table 2, Figure S3, S4). The activation energy of DNA duplex formation on thiol-ssDNA modified AuNPs is



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

similar to that of di-block DNA with polyA5 modified AuNPs. The regulation of the polyA length further reduced the activation energy from 24.40 to 14.48 kcal/mol/K. These results reinforced the lower energy barrier for DNA hybridization on programmable di-block DNA modified AuNPs.

Figure 4. (A) Kinetic of FAM-labeled target T1 dissociation from probe in solution (purple line) and on 13 nm AuNP in 0.1 M PBS at 319.15 K. (B) Kinetics of FAM-labeled target T1 dissociation from di-block probe on AuNPs with different polyA block length (5-40). The dashed lines are fits with first-order reaction equation. (c) Rate constant of kinetic of FAM-labeled target T1 dissociation from probe on 13 nm AuNPs in 0.1 M PBS at 319.15 K.

Table 3. Apparent activation energies determined from Arrenhenius plots for dissociation rate constant of duplex in solution and on AuNPs. System

Ead (kcal mol-1K-1)

AuNP-polyA5

36.33

AuNP-polyA10

39.95

AuNP-polyA15 AuNP-polyA20

40.11 41.76

AuNP-polyA30

43.40


Page 14 of 19

Page 15 of 19

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


AuNP-polyA40

44.36

AuNP-SH DNA

40.01

In parallel, we investigated the dissociation rates of DNA duplex on AuNPs by competitive strand-displacement with excessive unlabeled targets. The dissociation rate constants for duplex dissociation gradually decreased with the increase of polyA length, indicating that the stability of DNA duplex on AuNPs was programmably enhanced by increasing the polyA length (Figure 4). By analyzing the dissociation rate constants at different temperatures, we obtained the apparent activation energy (Ead in Table 3, Figure S5, S6). We found that activation energy was ~40-45 kcal/mol/K for dissociation of di-block duplex (polyA15~polyA40) on AuNPs, while the activation energy was ~40 kcal/mol/K for dissociation of thiolated duplex on AuNPs, indicating the enhanced stability of di-block duplex on AuNPs. Moreover, activation energies of di-block duplex on AuNPs were increased from ~36 kcal/mol/K to ~45 kcal/mol/K with the polyA length increased from 5 to 40 oligonucleotides, indicating that long polyA block enhanced the duplex structure on AuNPs. As a comparison, the DNA duplex with thiol anchoring exhibited a larger dissociation rate constant and demonstrated relatively poor stability in activation energy.

Conclusion In conclusion, we realized the programmable regulation of the surface density of DNA, DNA hybridization thermodynamics and kinetics on AuNPs. Our polyA-based DNA immobilization on AuNPs provides several advantages. First, natural nucleotides (adenines) are employed as anchoring moiety, which eliminates the additional modification of DNA and reduces the synthesis cost. Second, the



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

anchoring nucleotides (adenines) are highly oxidation resistant compared with some other anchoring moieties such as thiols. Hence, the longtime stability of DNA-AuNP nano-conjugates could be improved. Third, the surface density, hybridization thermodynamics and kinetics can be effectively regulated by tuning the polyA length. Fourth, beyond the single point anchoring, the adenines preferentially bind to gold surface and block nearly all the binding sites on the surface43, which eliminate the nonspecific interaction between other sequences and gold surface. Finally, our programmable assembly of DNA on AuNPs could provide excellent building blocks for bottom-up construction of nanoscale biosensors44-46. Furthermore, our results indicated that with the increased polyA length, the melting temperature was approaching that in solution. The increased polyA length would result in reduced DNA molecules on AuNP, which may decrease the absolute signal change in DNA hybridization analysis. However, the relative signal change can be used in DNA hybridization analysis.

AUTHOR INFORMATION Corresponding Author *E-mails: [email protected] ; [email protected]

Author Contributions †These authors contributed equally.

ACKNOWLEDGMENT This work was supported by the National Basic Research Program (973 Program 2013CB933802, 2012CB932600), NSFC (grant numbers 21305151, 21422508, 31470960, 21373260, 91123037) and Distinguished Scientist Fellowship Program of King Saud University.

SUPPORTING INFORMATION.


Page 16 of 19

Page 17 of 19

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


Supporting material on DNA surface density on AuNPs with various polyA block length, kinetics of DNA hybridization/dissociation in solution and on AuNPs and DNA sequences, is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES (1) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (2) Song, S. P.; Liang, Z. Q.; Zhang, J.; Wang, L. H.; Li, G. X.; Fan, C. H. Angew. Chem. In. Ed. 2009, 48, 8670-8674. (3) Cao, Y. W. C.; Jin, R. C.; Mirkin, C. A. Science 2002, 297, 1536-1540. (4) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757-1760. (5) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435-446. (6) Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. J. Am. Chem. Soc. 2007, 129, 15477-15481. (7) Tikhomirov, G.; Hoogland, S.; Lee, P. E.; Fischer, A.; Sargent, E. H.; Kelley, S. O. Nat. Nanotechnol. 2011, 6, 485-490. (8) Han, M. Y.; Gao, X. H.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631-635. (9) Schreiber, R.; Do, J.; Roller, E. M.; Zhang, T.; Schuller, V. J.; Nickels, P. C.; Feldmann, J.; Liedl, T. Nat. Nanotechnol. 2014, 9, 74-78. (10) Prigodich, A. E.; Seferos, D. S.; Massich, M. D.; Giljohann, D. A.; Lane, B. C.; Mirkin, C. A. ACS Nano 2009, 3, 2147-2152. (11) Giljohann, D. A.; Seferos, D. S.; Prigodich, A. E.; Patel, P. C.; Mirkin, C. A. J. Am. Chem. Soc. 2009, 131, 2072-2077. (12) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547-1562. (13) Sonnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A. P. Nat. Biotechnol. 2005, 23, 741-745. (14) Acuna, G. P.; Moller, F. M.; Holzmeister, P.; Beater, S.; Lalkens, B.; Tinnefeld, P. Science 2012, 338, 506-510. (15) Thacker, V. V.; Herrmann, L. O.; Sigle, D. O.; Zhang, T.; Liedl, T.; Baumberg, J. J.; Keyser, U. F. Nat. Commun. 2014, 5, 7. (16) Macfarlane, R. J.; Lee, B.; Jones, M. R.; Harris, N.; Schatz, G. C.; Mirkin, C. A. Science 2011, 334, 204-208. (17) Park, S. Y.; Lytton-Jean, A. K. R.; Lee, B.; Weigand, S.; Schatz, G. C.; Mirkin, C. A. Nature 2008, 451, 553-556. (18) Chou, L. Y. T.; Zagorovsky, K.; Chan, W. C. W. Nat. Nanotechnol. 2014, 9, 148-155. (19) Lin, M. H.; Pei, H.; Yang, F.; Fan, C. H.; Zuo, X. L. Adv. Mater. 2013, 25, 3490-3496. (20) Liu, B.; Ouyang, X. Y.; Chao, J.; Liu, H. J.; Zhao, Y.; Fan, C. H. Chin. J. Chem. 2014, 32, 137-141. (21) Tian, T.; Zhang, J. C.; Lei, H. Z.; Ying, Z.; Shi, J. Y.; Hu, J.; Huang, Q.; Fan, C. H.; Sun, Y. H. Nucl. Sci. Tech. 2015, 26, 6. (22) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768-11769. (23) Niazov, T.; Pavlov, V.; Xiao, Y.; Gill, R.; Willner, I. Nano Lett. 2004, 4, 1683-1687.



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

(24) Parbhu, A.; Lin, H.; Thimm, J.; Lal, R. Peptides 2002, 23, 1265-1270. (25) Thaxton, C. S.; Elghanian, R.; Thomas, A. D.; Stoeva, S. I.; Lee, J. S.; Smith, N. D.; Schaeffer, A. J.; Klocker, H.; Horninger, W.; Bartsch, G.; Mirkin, C. A. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 18437-18442. (26) Shen, W.; Deng, H. M.; Gao, Z. Q. J. Am. Chem. Soc. 2012, 134, 14678-14681. (27) Nam, J. M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 5932-5933. (28) Lytton-Jean, A. K. R.; Mirkin, C. A. J. Am. Chem. Soc. 2005, 127, 12754-12755. (29) Chen, C. L.; Wang, W. J.; Ge, J.; Zhao, X. S. Nucleic Acids Res. 2009, 37, 3756-3765. (30) Xu, J.; Craig, S. L. J. Am. Chem. Soc. 2005, 127, 13227-13231. (31) Furst, A. L.; Hill, M. G.; Barton, J. K. Langmuir 2013, 29, 16141-16149. (32) Josephs, E. A.; Ye, T. J. Am. Chem. Soc. 2012, 134, 10021-10030. (33) Pei, H.; Li, F.; Wan, Y.; Wei, M.; Liu, H. J.; Su, Y.; Chen, N.; Huang, Q.; Fan, C. H. J. Am. Chem. Soc. 2012, 134, 11876-11879. (34) Yao, G. B.; Pei, H.; Li, J.; Zhao, Y.; Zhu, D.; Zhang, Y. N.; Lin, Y. F.; Huang, Q.; Fan, C. H. NPG Asia Mater. 2015, 7, 7. (35) Zhu, D.; Pei, H.; Chao, J.; Su, S.; Aldalbahi, A.; Rahaman, M.; Wang, L. H.; Wang, L. H.; Huang, W.; Fan, C. H.; Zuo, X. L. Nanoscale 2015, 7, 18671-18676. (36) Morrison, L. E.; Stols, L. M. Biochemistry 1993, 32, 3095-3104. (37) Hurst, S. J.; Lytton-Jean, A. K. R.; Mirkin, C. A. Anal. Chem. 2006, 78, 8313-8318. (38) Zhu, D.; Chao, J.; Pei, H.; Zuo, X. L.; Huang, Q.; Wang, L. H.; Huang, W.; Fan, C. H. ACS Appl. Mater. Interfaces 2015, 7, 11047-11052. (39) Gong, P.; Levicky, R. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 5301-5306. (40) Gao, Y.; Wolf, L. K.; Georgiadis, R. M. Nucleic Acids Res. 2006, 34, 3370-3377. (41) Pei, H.; Lu, N.; Wen, Y. L.; Song, S. P.; Liu, Y.; Yan, H.; Fan, C. H. Adv. Mater. 2010, 22, 4754-4758. (42) Pei, H.; Zuo, X. L.; Pan, D.; Shi, J. Y.; Huang, Q.; Fan, C. H. NPG Asia Mater. 2013, 5, 9. (43) Opdahl, A.; Petrovykh, D. Y.; Kimura-Suda, H.; Tarlov, M. J.; Whitman, L. J. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 9-14. (44) Wilner, O. I.; Willner, I. Chem. Rev. 2012, 112, 2528-2556. (45) Willner, I.; Shlyahovsky, B.; Zayats, M.; Willner, B. Chem. Soc. Rev. 2008, 37, 1153-1165. (46) Pei, H.; Zuo, X. L.; Zhu, D.; Huang, Q.; Fan, C. H. Acc. Chem. Res. 2014, 47, 550-559.


Page 18 of 19

Page 19 of 19

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


For TOC only:


PolyA-Mediated DNA Assembly on Gold Nanoparticles for

Recommend Documents