(DNA) Oligomers - Army Research Laboratory

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PROGRAM ELEMENT NUMBER. 6. AUTHOR(S) ... TELEPHONE NUMBER (Include area code). (410) 306- ... Figure 1. ss-DNA Ba813 with the 5' end at the top.
Experimental and Theoretical Studies to Characterize the Fundamental Interactions Between Deoxyribonucleic Acid (DNA) Oligomers and Gold (Au) Nanoparticles by Molleshree Karna, Radhakrishnan Balu, Govind Mallick, and Mark Griep

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September 2011

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Army Research Laboratory Aberdeen Proving Ground MD 21005-5069

ARL-TR-5765

September 2011

Experimental and Theoretical Studies to Characterize the Fundamental Interactions Between Deoxyribonucleic Acid (DNA) Oligomers and Gold (Au) Nanoparticles Molleshree Karna Science and Mathematics Academy, Aberdeen High School

and Radhakrishnan Balu, Govind Mallick, and Mark Griep Weapons and Materials Research Directorate, ARL

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Summary

16 June to 9 December 2010

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Experimental and Theoretical Studies to Characterize the Fundamental Interactions Between Deoxyribonucleic Acid (DNA) Oligomers and Gold (Au) Nanoparticles

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Molleshree Karna, Radhakrishnan Balu, Govind Mallick, and Mark Griep

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U.S. Army Research Laboratory ATTN: RDRL-WMB-D Aberdeen Proving Ground MD 21005-5069

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14. ABSTRACT

A combined experimental and theoretical investigation of mixtures of gold (Au) nanoparticles (NPs) and single stranded deoxyribonucleic acid (DNA) (ss-DNA) were carried out to characterize the nature of their interaction. Au NPs are found to bind with ss-DNA under mildly acidic conditions. Atomic force microscopy (AFM) images and ultraviolet-visible (UV-Vis) spectroscopy of the mixture provided evidence for the binding of Au NPs with ss-DNA. The theoretical calculations provided a possible charge transfer pathway from the DNA base guanine to Au atoms, thus characterizing the interaction as electrostatic. The calculations outline the possible effect of the presence of other bases to guanine-mediated charge transfer. Specifically, the presence of an adenine base alters the charge localization at the guanine base, thus preventing charge transfer to the NPs. 15. SUBJECT TERMS

Nanoparticles, AFM, UV-Vis, QD, DNA 17. LIMITATION OF ABSTRACT

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Contents

List of Figures

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1.

Introduction and Background

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2.

Materials, Methods and Computational Details

2

3.

2.1

Experimental Methodology .............................................................................................2

2.2

Theoretical Calculations ..................................................................................................2

Results and Discussion

3

3.1

Spectral Analysis .............................................................................................................3

3.2

Atomic Force Microscopic (AFM) Analysis...................................................................3

3.3

Quantum Chemical Analysis ...........................................................................................5

4.

Summary and Conclusions

8

5.

References

9

List of Symbols, Abbreviations, and Acronyms

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Distribution List

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List of Figures Figure 1. ss-DNA Ba813 with the 5' end at the top. The guanine bases are colored in yellow. .....1 Figure 2. UV-Vis absorption spectrum of the Au NP and NP-DNA sample, with a peak around 521 nm for both samples................................................................................................3 Figure 3. Non-contact mode AFM image of (a) Au NPs (scan size: 5 μm) and (b) DNA (scan size: 2 μm) on cleaved muscovite mica. The Au NPs are very concentrated and vary in size between 30 to 100 nm wide. The DNA branches off of an agglomeration and is about 80 nm wide.................................................................................................................................4 Figure 4. Non-contact mode AFM image of the Au NP-DNA mixture (scan sizes: 10 and 5 μm) on cleaved muscovite mica. ...............................................................................................4 Figure 5. QD-DNA at 250x250 nm2 resolution. ..............................................................................5 Figure 6. Oligomer GMP. ................................................................................................................5 Figure 7. Oligomer GMP with the Au cluster..................................................................................6 Figure 8. HOMO of the Au3-GMP showing charge transfer from the guanine base to the Au cluster. ........................................................................................................................................6 Figure 9. HOMO of 5'-G-T-C-A-3'..................................................................................................7

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1. Introduction and Background Semiconductor quantum dots (QDs) and metal nanoparticles (NPs) have attracted a great deal of attention in biology due to their application as fluorescent labels and sensors (1–4). The optical properties of NPs allow them to be effective imaging agents. However, size-dependent NPs have the potential to be used as more than just sensors and labels (5–9). Their biological sensing abilities include identifying target deoxyribonucleic acid (DNA) through a linker (1, 3), followed by color change and/or electrical signaling. In addition, the self assembly of NPs themselves can be carried out with the help of DNA templates. When NPs are mixed with single stranded DNA (ss-DNA), the hybridization of single strands can lead to assembly of NPs attached to DNA. Advances in the design of DNA origami has enabled hybrid nano-DNA assemblies (15) and their functionalizations. Thus, it is essential to understand the fundamental interaction between DNA strands and NPs at the base level. Recently, there has also been a series of theoretical investigations on gold (Au) DNA interactions. The studies (10, 11) consist of models containing nucleobases, that is, nucleotides without the ribose sugar and phosphate groups, with a few Au atoms placed in proximity of the electronegative atoms of the bases. In the present study, experimental and theoretical methods were used to explore the fundamental interactions between NPs and DNA. Atomic force microscopy (AFM) and ultraviolet-visible (UV-Vis) spectroscopy show interaction between colloidal Au NPs and a 30-base sequence ss-DNA (figure 1), whereas quantum mechanical density functional theory (DFT) calculations revealed the high affinity of guanine monophosphate (GMP) with NPs compared to the other nucleic acid bases of DNA.

Figure 1. ss-DNA Ba813 with the 5' end at the top. The guanine bases are colored in yellow.

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2. Materials, Methods and Computational Details 2.1

Experimental Methodology

Au NPs were synthesized using the sodium citrate reduction method (10). A volume of 100 mL of 0.01% aurochloric acid (HAuCl4) was heated to boiling. Then, 1.9 mL of 1% trisodium citrate dihydrate (Na3C6H5O7 . 2H2O) was added to the boiling solution under constant stirring. The solution was removed from heat 15 min after the trisodium citrate was added, and the cooling solution was supplemented with 120 µL of 1M sodium chloride (NaCl) to end the reaction. This procedure provides fairly uniform size Au NPs capped by citrate ions (12, 13). The DNA solution was prepared using the following method. First, 14.74 mL of tris-borateEDTA, with a pH of 8.3 buffer, was used to dissolve a 1474-nmol ss-DNA oligomer (Ba813 Target 30, 30 base pair, 5' CAT TAA CGA GTT ACT CAA TGA GTC TTT) obtained from Integrated DNA Technologies. The solution was agitated to dissolve the DNA into the buffer. To prepare the DNA to be viewed under the Autoprobe AFM, a 100-µM DNA solution and a 100-µM CaCl2 solution, used to ensure adsorption to the substrate (13), were mixed at a 1:1 volume ratio (1 mL of each). Then, 50 µL of the 1:1 solution was spin-coated onto a muscovite mica substrate that was cleaved and rinsed with CaCl2 at a rate of 1500 rpm for 6 min. The NPs were centrifuged at 8500 x g for 30 min. The supernatant was removed, and the pellet was diluted with 10-mL tris-borate-EDTA buffer. Subsequently, 50 µL of the NP solution was spin-coated onto a cleaved muscovite mica substrate at a rate of 1500 rpm for 6 min. A NP/DNA solution was mixed at a 1:1 ratio. Then, 2 mL of each diluted solution was put into a vial rinsed with ethanol. A 50-µL volume of the 1:1 solution was spin-coated onto a muscovite mica substrate that was cleaved and rinsed with calcium chloride (CaCl2) (14) at a rate of 1500 rpm for 6 min. The spin-coated substrates for each sample were probed under an AFM using the noncontact mode. The absorption spectrum for each of the sample solutions was obtained using a photonics-charge-coupled device (CCD) array UV-Vis spectrophotometer, and the emission and excitation spectra were obtained using a Horiba Fluoromax-3 spectrofluorometer. 2.2

Theoretical Calculations

Ab initio quantum calculations were performed on the Au-ss-DNA system with GMP as the starting point of the model. Subsequently, calculations were performed by adding adenine, cytosine, and thymine bases in the GMP monomer. To represent a realistic ss-DNA, the final oligomer contains all four DNA bases separated by phosphate ester. The size of the oligomer was extended from one to four, considering many possible combinations of bases. In the case of dimers, both guanine and adenine bases were considered. For trimers, work with several combinations of G-A-C, G-A-T, G-C-T, and G-C-T oligomers was performed. Finally, all four bases in two different single stranded configurations were analyzed. One configuration with 2

guanine and adenine bases adjacent to each other and the other where the bases were kept far apart at the 5' and 3' ends, respectively. To avoid any possible bias, the guanine base was moved to different positions of the oligomer in the configurations considered. The Au NP was nominally represented by a 3-atom cluster. The size of the cluster was left small to manage computational cost. However, the size of Au cluster is not considered to affect the findings, since our focus was on understanding the nature of the interaction between the Au and the nucleic acid bases. Geometries of the systems studied were relaxed at the DFT level using hybrid functional B3LYP without applying symmetry constraints. The standard 6-32G(d) basis set was used for all atoms except for Au, for which the LANL2DZ effective core potential (ECP) was used. All calculations were carried out using the Gaussian 09 package.

3. Results and Discussion 3.1

Spectral Analysis

UV-Vis spectroscopy (figure 2) was used to determine the absorption, emission, and excitation of the samples. The Au QDs showed peaks at a wavelength of 521 nm for absorption. The NPDNA mixture showed a broad peak at a wavelength of 521 nm for absorption. The broad absorption peak of the QDs reveals that the solution does not contain QDs of uniform size.

Figure 2. UV-Vis absorption spectrum of the Au NP and NP-DNA sample, with a peak around 521 nm for both samples.

3.2

Atomic Force Microscopic (AFM) Analysis

AFM was used to probe the morphology and surface property of the NPs, DNA, and the NPDNA complex on the mica substrate (figure 3 through 5). The NPs’ widths ranged between 30 to 100 nm, and their average height was about 3 nm, suggesting clustering or agglomeration of small NPs into larger particles. The DNA sample showed individual strands coming off of larger pieces of DNA agglomerations; the DNA was 1 nm high on average and 80 nm wide on average. 3

The NP-DNA sample showed both QDs and DNA oligomer strands. The DNA’s average height is 8 nm; the NPs’ dimensions are on average 10 nm high and 300 nm wide. Though both DNA and NPs are visible in the images and seem to be physically close to each other, it is unclear whether the two are interacting near the guanine bases. The DNA strands seem to occur more frequently than the NPs.

Figure 3. Non-contact mode AFM image of (a) Au NPs (scan size: 5 μm) and (b) DNA (scan size: 2 μm) on cleaved muscovite mica. The Au NPs are very concentrated and vary in size between 30 to 100 nm wide. The DNA branches off of an agglomeration and is about 80 nm wide.

Figure 4. Non-contact mode AFM image of the Au NP-DNA mixture (scan sizes: 10 and 5 μm) on cleaved muscovite mica.

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Figure 5. QD-DNA at 250x250 nm2 resolution.

3.3

Quantum Chemical Analysis

Analysis started with a ss-DNA oligomer containing GMP (figure 6) and optimized its geometry in a gas phase, as shown in figure 7. The Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) frontier orbitals of the relaxed structure were characterized, yielding qualitatively similar orbitals to the ones obtained in the previous studies (5). A total of three Au atoms were added closer to the N7 of the guanine base (as shown in figure 8) and plotted the frontier orbitals in figure 9. As can be seen from figure 9, the charge localized on guanine base and subsequently transferred to Au atoms confirming the results from previous studies (5).

Figure 6. Oligomer GMP.

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Figure 7. Oligomer GMP with the Au cluster.

Figure 8. HOMO of the Au3-GMP showing charge transfer from the guanine base to the Au cluster.

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Figure 9. HOMO of 5'-G-T-C-A-3'.

The model was extended by adding an adenine base building a ss-DNA oligmer with a guanine base at the 5' position and the adenine base at the 3' position. The HOMO and LUMO frontier orbitals of the oligomer share the same charecterestics of the G-C pair studied by the Shukla et al. (6). That is, the HOMO is located on the adenine base and the LUMO is located on the guanine base. In the previous study (5) of C-G base pairs, the HOMO was located on cytosine and LUMO was located on guanine. Additional Au atoms were added to the single stranded dimer by placing the atoms closer to guanine base and plotted the frontier orbitals. The charge transfer from the ss-DNA oligomer to the Au atoms was absent and required an excitation for the transfer to effect. This shows the effect of the presence of an adenine base on localization charge and the consequent transfer to Au on a guanine base. All four combinations of trimeric oligomers were considered and it was found that guanine base is the prefered base for charge localization. Initial focus was on the trimer 5'G-A-C-3' to compute the frontier orbitals. In HOMO the charge was localized on guanine and in LUMO the localization was on cytosine and guanine. When considering the combination 5'-T-G-C-3', the charge localization for HOMO and LUMO occurred exclusively on guanine. For the combination of 5'-G-A-T-3', the charge localization was found to be on guanine for both HOMO and LUMO. The final combination of trimer excluding guanine was 5'-A-C-T-3' and the charge localization was not on adenine but rather on thymine for HOMO and cytosine for LUMO. It has become clear that guanine is the prefered base for charge localization and adenine presence sometimes influences that localization even though the charge does not localize on adenine in the absence of guanine. 7

Finally, a model was built all four bases and performed the HOMO and LUMO calculations. The first tetramer ss-DNA had the sequence 5'-C-A-G-T-3'. The localization of charge occurred on both guanine and adenine for HOMO and only on guanine for LUMO. The other tetramer considered had the configuration of 5'-G-T-C-A-3'. In this configuration, the presence of adenine residue clearly interfered with guanine and the charge localized on adenine for HOMO, as shown in figure 9, and on cytosine for LUMO.

4. Summary and Conclusions The linkages of NPs with specific sites of DNA as revealed by the morphological AFM image in figure 5 indicate the binding of Au NPs to DNA. The UV-Vis spectral analysis, performed at the excitation energy of 250 nm, showed the emission of DNA and Au at the expected wavelengths of 381 and 461 nm, respectively (not shown). However, the emission spectrum (figure 2) of the mixture showed a broad range, but there were two distinct peaks at 396 and 469 nm, indicating a strong interactions between Au and DNA with modification in their individual electronic structures. The possible reasons of positive shift in the wavelengths are unknown and requires further investigation. Quantum mechanical calculations showed that Au NPs naturally link with ss-DNA. The results indicate that among the nucleic acid bases guanine interacts with Au atoms more than other bases and the interaction is affected in the presence of adenine. One of the key features of the interactions is that after binding the Au clusters, there is a net charge transfer from the DNA base to the Au cluster.

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5. References 1. Algar, W. R. et al. Anal. Bioanal. Chem. 2008, 391, 1609. 2. Tran, P. T. et al. Phys. Stat. Sol. 2002, 229, 427. 3. Lee, J. et al. Chem Phys Chem. 2009, 10, 806. 4. Cady, N. C. et al. Mol. Cell. Probes 2007, 21, 116. 5. Medintz, I. L. et al. Nat. Mat. 2003, 2, 630–638. 6. Medintz, I. L. et al. Proc. Natl. Acad. Sci. 2004, 101, 9612–9617. 7. Deniz, A. A. et al. Proc. Natl. Acad. Sci. 1999, 96, 3670–3675. 8. Wang, Y. et al. Mat. Today 2005, 8, 20–31. 9. Bauer, G. et al. Inst. of Phys. Publ. 2003, 14, 1229–1311. 10. Sharma, P. et al. J. Theor. Comput. 2008, 113, 2301. 11. Shukla, M. K. et al. J. Phys. Chem. 2009, 113, 3960. 12. Sawant, B. et al. to be published. 13. Mcfarland, A. D. et al. J. Chem. Educ. 2004, 81, 544A. 14. Cheng, H. et al. Biophys. J. 2005, 90, 1164–1174. 15. Bellido, E. P. et al. Nanotechnology 2009, 20, 175102–175107.

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List of Symbols, Abbreviations, and Acronyms AFM

atomic force microscopy

Au

gold

CaCl2

calcium chloride

CCD

charge-coupled device

DFT

density functional theory

DNA

deoxyribonucleic acid

ECP

effective core potential

GMP

guanine monophosphate

HAuCl4

aurochloric acid

HOMO

Highest Occupied Molecular Orbital

LUMO

Lowest Unoccupied Molecular Orbital

Na3C6H5O7 . 2H2O

trisodium citrate dihydrate

NaCl

sodium chloride

NPs

nanoparticles

QDs

quantum dots

ss

single stranded

UV

ultraviolet

Vis

visible

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No. of Copies 1 ELEC

Organization ADMNSTR DEFNS TECHL INFO CTR ATTN DTIC OCP 8725 JOHN J KINGMAN RD STE 0944 FT BELVOIR VA 22060-6218

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US ARMY RSRCH LAB ATTN IMNE ALC HRR MAIL & RECORDS MGMT ATTN RDRL CIO LL TECHL LIB ATTN RDRL CIO MT TECHL PUB ADELPHI MD 20783-1197

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US ARMY RSRCH LAB ATTN RDRL WML B MOLLESHREE KARNA GOVIND MALLICK ATTN RDRL WMM A RADHAKRISHNAN BALU MARK GRIEP (5 COPIES) BLDG 4600 ABERDEEN PROVING GROUND MD 21005-5066

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